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，但它们分化成200多种不同的细胞类型，执行特定的功能。这些不同类型的细胞以精确的空间模式发育，形成胚胎的身体计划。几十年的遗传和生物化学研究表明，细胞通过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向每个细胞传递位置信息至关重要，因此它可以正确地进行专业化。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，信号解释和专门化的过程如何发生仍然不清楚。这种挑战对于在子宫内发育的哺乳动物胚胎来说尤其严重。人类胚胎干细胞（hESCs）代表了这些挑战的潜在解决方案，因为它们能够在培养皿中遵循发育程序，能够在一定程度上阐明人类发育，否则是不可能的。该项目结合了在特定空间模式下生长的hESCs的实验研究和数学建模，以剖析细胞在发育的早期阶段如何解释信号并在空间模式下分化。与此研究计划相结合的是一系列旨在培养下一代科学家在生物科学领域进行跨学科定量研究的教育活动。计划的活动包括拓展到K-12学生，针对高中先修课程教师的教育，以及创新的本科和研究生课程。在莱斯大学成功推出后，这些课程将通过Coursera在线平台广泛传播。由于在子宫内观察和操纵胚胎的困难，哺乳动物发育过程中图案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，其中使用微图案技术在受控几何形状中生长的hESC菌落形成图案。该模式由添加到培养基中的诱导信号（BMP4）以及细胞间的旁分泌信号（已被识别为Wnt和激活素-节点信号）组合而成。该项目将使用该系统定量地解构哺乳动物胚胎早期模式的机制。首先，类似的微图技术将用于分离极小的hESCs菌落（1-10个细胞）。带有单个细胞的菌落将用于直接测量对感应信号的响应，而不受邻近细胞的干扰。有两个细胞的菌落将被用来理解在一个简化的系统中对旁分泌信号的反应，而那些有更多细胞的菌落将被用来测量细胞如何整合来自多个邻居的信号。这些小菌落的结果将用于建立数学模型，能够预测数千个细胞组成的更大菌落的模式，这些细胞显示出胚胎命运的全部谱。这些模型将通过使用BMP、Wnt和Nodal信号通路活性的活细胞成像报告因子以及细胞命运报告因子的实验得到验证和进一步发展。这些动态观察只可能在体外发育系统中进行，并将用于将每个细胞中的信号动力学与其最终采用的命运联系起来。综上所述，这些结果将为早期哺乳动物的发育提供前所未有的理解，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.