CAREER: Understanding human embryonic development from the bottom up

发育中的人类胚胎的细胞都携带相同的遗传物质，但它们专门形成200多种不同的细胞类型，执行特定的功能。这些不同的细胞类型以精确的空间模式发育，形成了胚胎的身体平面。数十年的遗传和生化研究表明，细胞使用分泌的信号分子进行交流，这些分子在细胞内激活的信号通路对于将位置信息传递给每个细胞，使其正确专门化是必不可少的。虽然这些途径的许多组成部分已经被发现，但由于观察和干扰胚胎发育的困难，信号解释和特化的过程如何发生仍然不清楚。对于在子宫内发育的哺乳动物胚胎来说，这一挑战尤为严峻。人类胚胎干细胞(HESCs)代表了这些挑战的潜在解决方案，因为它们能够遵循培养皿中的发育程序，能够在其他情况下不可能的程度上阐明人类的发育。该项目结合了对在特定空间模式下生长的hESCs的实验研究和数学建模，以剖析细胞如何在发育的最早阶段解释信号和在空间模式中进行分化。与这一研究计划相结合的是一系列旨在培训下一代科学家在生物科学中进行跨学科、定量研究的教育活动。计划的活动包括面向K-12学生的外展，针对高中高级安置教师的教育，以及创新的本科生和研究生课程。在莱斯大学成功引入这些课程后，这些课程将通过Coursera在线平台广泛分发。由于观察和操纵子宫内胚胎的困难，哺乳动物发育过程中模式形成的机制研究具有挑战性。该项目利用了最近开发的体外系统，在该系统中，使用微图案化技术在生长在受控几何形状的hESC克隆中形成图案。这些模式是由添加到培养基中的诱导信号(BMP4)以及细胞之间的旁分泌信号(已被鉴定为Wnt和Activin-Nodal信号)的组合形成的。该项目将使用这一系统来定量解构哺乳动物胚胎早期图案形成的机制。首先，类似的微图案化技术将被用于分离极小的hESCs克隆(1-10个细胞)。单个细胞的菌落将被用来直接测量对诱导信号的响应，而不受邻近细胞的干扰。在一个简化的系统中，有两个细胞的克隆将被用来了解对旁分泌信号的反应，而那些拥有更多细胞的克隆将被用来测量细胞如何整合来自多个邻居的信号。这些小集落的结果将被用来创建数学模型，能够预测由数千个细胞组成的大集落的模式，这些细胞展示了胚胎命运的全部光谱。这些模型将通过使用活细胞成像记者对BMP、Wnt和Nodal信号通路的活性以及细胞命运的记者进行验证和进一步开发。这些动态观察只有在体外发育系统中才有可能，并将被用来将每个单个细胞中的信号动力学与它最终采用的命运联系起来。综上所述，这些结果将提供对早期哺乳动物发育的前所未有的理解，并揭示旁分泌信号和体外组织自组织的原理。

The cells of the developing human embryo all carry the same genetic material, but they specialize into more than 200 different cell types that perform specific functions. These different cell types develop in precise spatial patterns that form the body plan of the embryo. Decades of genetic and biochemical studies have revealed that cells communicate using secreted signaling molecules, and that the signaling pathways activated inside the cell by these molecules are essential for conveying positional information to each cell, so that it specializes correctly. While many of the components of these pathways have been discovered, how the processes of signal interpretation and specialization occur has remained obscure due to the difficulty of observing and perturbing embryonic development. This challenge is particularly acute for mammalian embryos that develop in utero. Human embryonic stem cells (hESCs) represent a potential solution to these challenges, as they are capable of following developmental programs in a culture dish, enabling elucidation of human development to a degree that is otherwise impossible. This project combines experimental studies of hESCs grown in particular spatial patterns and mathematical modeling to dissect how cells interpret signals and differentiate in spatial patterns during the earliest stages of development. Coupled with this research program is a spectrum of educational activities aimed at training the next generation of scientists to perform interdisciplinary, quantitative research in the biological sciences. Planned activities include outreach to K-12 students, education aimed at high school advanced placement teachers, and innovative undergraduate and graduate courses. Following successful introduction at Rice University, these courses will be widely distributed through the Coursera online platform.Mechanistic studies of pattern formation during mammalian development are challenging due the difficulty of observing and manipulating the embryo in utero. This project takes advantage of a recently developed in vitro system in which patterns form in hESC colonies grown in controlled geometries using micropatterning technology. The patterns form from the combination of an inductive signal added to the culture medium (BMP4) as well as paracrine signals between cells that have been identified as Wnt and Activin-Nodal signals. The project will use this system to quantitatively deconstruct the mechanisms of early patterning in the mammalian embryo. First, similar micropatterning technology will be used to isolate extremely small colonies of hESCs (1-10 cells). Colonies with a single cell will be used to directly measure the response to the inductive signal without interference from neighboring cells. Colonies with two cells will be used to understand the response to the paracrine signals in a simplified system, while those with larger number of cells will be used to measure how cells integrate signals from multiple neighbors. Results from these small colonies will be used to create mathematical models capable of predicting patterning in larger colonies of thousands of cells that display the full spectrum of embryonic fates. These models will be validated and further developed by experiments using live-cell imaging reporters for the activity of the BMP, Wnt, and Nodal signaling pathways together with reporters for cell fates. These dynamic observations are only possible in the in vitro development system, and will be used to correlate the dynamics of signaling in each individual cell with the fate it ultimately adopts. Taken together, these results will provide unprecedented understanding of early mammalian development and reveal principles of paracrine signaling and in vitro tissue self-organization.