Developing organoid model to study active folding in a human genetic context

摘要 器官的形状对有机体的正常运作至关重要。以神经管为例，早期形成 缺陷可能会对身体造成毁灭性的后果。胚胎发育中的一个基本构建原则是 首先将细胞组织成简单的结构，如围绕管腔的闭合薄片。然后细胞会经历 协调良好的程序，以重新配置组织形状，或折叠出片材的平面。在平面流动中 在收敛延伸过程中，延长一条轴，同时缩短另一条轴。对比鲜明的折叠效果 改变组织形式，赋予器官以其特有的形状。在神经管闭合中，一开始是平的 一张单元格经历两个连续的弯曲步骤，折叠成围绕着单个单元格的圆柱管 流明。一大堆工作揭示了关于命运决定的基本见解。然而，机制 控制形状，特别是在人类早期发育的背景下折叠，仍然知之甚少。 在细胞水平上，遗传模式协调远距离的行为，指示活跃的力量 向下折叠。在提取力量如何折叠组织的物理图像之前，我们需要识别行为 导致折叠，并以协调为特征。有机化合物为研究早期发育提供了希望， 在严格控制的环境和人类遗传背景下的疾病和再生。然而， 由于不规则的图案和形状，技术限制阻碍了进展。这项建议旨在 开发一种新的策略来设计健壮的模型来研究人类器官发生的基本机制。 使用微图案，我们成功地产生了具有固定大小、形状和形状的高度可重复性的有机化合物 形成动力学。我们的方法以扁平的矩形板材为特色，这些板材可以自发地从 平面，并沿着短维度无缝闭合，生成管子。由此产生的平台是高的 吞吐量，与实时成像兼容，非常适合定量数据分析策略。此设置 概述了动物模型系统中神经管关闭的主要步骤，如铰链的形成和 拉链机构与肌动球蛋白电缆伴随关闭。在这里，我们的目标是将现有协议调整为 我们的平台1)诱导神经上皮细胞的形成，并更接近地模拟神经管的形成。然后我们 目的2)建立闭合过程中观察到的细胞行为定量图谱。最后，我们的目标是进入 新的领域，并利用微流控技术生成横跨有机管的解剖轴。我们的方法 以神经管为例，开辟了一条通向人类器官发生受控模型的新途径。

ABSTRACT Organ shape is critical for the organism to function properly. For the example neural tube, early formation defects may have devastating consequences to the body. A fundamental building principal in embryogenesis is first organizing cells into simple structures, such as a closed sheet surrounding a lumen. Cells then undergo well-concerted programs to reconfigure tissue shape, or fold out of the plane of the sheet. In plane flows elongate one axis while shortening another during convergent extension. Folding in contrast dramatically changes tissue form, to endow the organ with its characteristic shape. In neural tube closure, an initially flat sheet of cells undergoes two sequential bending steps to fold up into a cylindrical tube surrounding a single lumen. A large body of work uncovered fundamental insights on fate determination. Yet, mechanisms controlling shape, particularly folding in the context of early human development, remain poorly understood. At the cellular level genetic patterning coordinates behaviors over long distances to instruct active forces that underlie folding. Before distilling a physical picture of how forces fold tissue, we need to identify behaviors leading to folding, and characterize coordination. Organoids harbor promise for studying early development, disease and regeneration in tightly controlled environments and human genetic background. However, progress is hampered by technical limitations, due to irregular patterning, and shape. This proposal seeks to develop a new strategy for designing robust models to study basic mechanisms of human organogenesis. Using micro patterning we successfully generated highly reproducible organoids with set size, shape, and formation kinetics. Our approach features flat rectangular shaped sheets that spontaneously fold out of the plane, and seamlessly close along the short dimension, generating a tube. The resulting platform is high throughput, compatible with live imaging, and well suited for quantitative data analysis strategies. This setup recapitulates major steps during neural tube closure in animal model systems, such as hinge formation and a zippering mechanism with actomyosin cable accompanying closure. Here we aim to adapt existing protocols to our platform to 1) induce formation of neuroepithelium and more closely model neural tube formation. We then aim to 2) assemble a quantitative atlas of cell behaviors observed during closure. Finally, we aim to 3) enter new territory and generate anatomical axes spanning the organoid tube using microfluidics. Our approach opens up a new path towards controlled models of human organogenesis, using the neural tube as example.