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

基本信息

批准号：
10316204
负责人：
Aryeh Warmflash
金额：
$ 32.27万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10316204
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 directed differentiation experimental study gastrulation human embryonic stem cell inhibitor loss of function mathematical model novel paracrine relating to nervous system response small molecule stem cells temporal measurement tool

项目摘要

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.

项目摘要 WNT途径在胚胎发育和疾病中起着至关重要的作用。在早期开发，wnt信号传导是需要进行过度和肠系膜分化的，并且稍后，为了分化外胚层内的神经rest细胞。而信号转导的详细生物化学已被广泛研究，少得多关于途径活动的动力学。我们已经使用CRISPR-CAS9基因组工程在两个人类胚胎干细胞（HESC）中创建一个新的记者，用于Wnt途径动力学和人类癌细胞以暂时分钟的暂时分辨率。使用此工具，我们发现，Wnt配体的刺激会产生信号传导的短暂定位效应子β-catenin对细胞核。正如以前的工作所表明的那样，这令人惊讶，我们已经证实了我们的记者，Wnt稳定地升高了总β-catenin水平信号。在这里，我们建议将自动流体控制的组合进行操作配体动力学，Wnt途径和细胞脂肪记者的定量测量以及数学建模以剖析配体输入之间的关系，信号转导 β-catenin和由此产生的细胞脂肪决策。我们将检查内源性Wnt的作用使用新型的hESC培养平台中的人类外胚层模式传导。在这个系统中，使用微图案技术在受控的几何形状中生长，与外皮命运，然后用配体BMP4处理。我们已经证明了BMP4响应导致菌落边界附近的表皮命运，并引起次级内源性 Wnt信号导致菌落内特定半径的神经rest分化。细胞在殖民地的中心没有收到这些信号，并采用神经命运。我们将结合该测定的WNT和BMP途径活动和细胞脂肪标记的测量测定了解提供的BMP信号与诱导的Wnt信号之间的相互作用如何创建外止细胞命运的自组织模式。该项目将提供必不可少的有关WNT途径动态的信息，这对于理解其在胚胎中的作用至关重要开发，以指示分化协议来利用它，并在情况下进行规避 wnt信号传导驱动癌症的生长。此外，了解Wnt在体外外胚层模式测定将揭示产生自组织组织的原则体外，再生医学的核心目标。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Stem-cell-based embryo models for fundamental research and translation.

用于基础研究和翻译的基于干细胞的胚胎模型。

DOI：
10.1038/s41563-020-00829-9
发表时间：
2021-03
期刊：
Nature materials
影响因子：
41.2
作者：
Fu J;Warmflash A;Lutolf MP
通讯作者：
Lutolf MP

Self-organized signaling in stem cell models of embryos.

DOI：
10.1016/j.stemcr.2021.03.020
发表时间：
2021-05-11
期刊：
Stem cell reports
影响因子：
5.9
作者：
Liu L;Warmflash A
通讯作者：
Warmflash A

Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids.

DOI：
10.1038/s41467-022-28149-3
发表时间：
2022-01-25
期刊：
Nature communications
影响因子：
16.6
作者：
Liu L;Nemashkalo A;Rezende L;Jung JY;Chhabra S;Guerra MC;Heemskerk I;Warmflash A
通讯作者：
Warmflash A

Insights into mammalian morphogen dynamics from embryonic stem cell systems.