CAREER: Elucidating spatial and epigenetic regulation of gene expression during human development using photopatterning and single-cell multiomics

复杂的生物有机体是由单个细胞产生的。然而，我们对这个单细胞如何分裂和分化成构成生物体内不同组织的所有特化细胞的理解是相当有限的。本研究的目的是了解细胞在组织中的空间位置及其表观遗传状态如何在人类早期发育过程中调节基因表达。细胞的功能受到细胞在组织中的位置及其内在表观遗传环境的严格控制。虽然单细胞测序通过测量单细胞内的转录组和表观基因组景观彻底改变了我们对哺乳动物系统的理解，但使用现有方法会丢失空间信息。为了更好地理解空间组织如何调节单个细胞中的基因表达，PI建议使用光敏寡核苷酸在发育过程中光学标记细胞的空间位置。该空间信息将与单细胞mRNA测序和单细胞表观遗传特征相结合。总的来说，这些技术将绘制空间和表观遗传决定因素如何在早期发育过程中控制人类原始生殖细胞的规格。研究目标产生的数据将用于一个多层次的综合教育项目，其中包括针对高中生的计算基因组学训练营，针对本科生的夏季湿实验室实习，以及针对高中生介绍基因调控概念的移动应用程序游戏。为了全面研究空间组织如何调节单个细胞的基因表达，PI将设计胆固醇标记的光敏寡核苷酸（PSO），将其整合到细胞膜中，从而在组织解离和单细胞mRNA测序之前对每个细胞的位置进行光学“印记”。定量mRNA和单个细胞中pso的相对降解将能够以高分辨率绘制空间单细胞转录组。此外，PI将开发空间分辨单细胞多组学技术，同时对来自同一细胞的转录组以及不同的表观遗传特征进行测序，以直接关联DNA甲基化、DNA可及性和组蛋白标记如何在不同的空间背景下调节基因表达。此外，通过采用迭代轮次的光学标记，他们将通过终点单细胞多组学测序捕捉细胞迁移，以深入了解组织形态发生如何影响细胞表型。这些转化方法将用于具体研究形态梯度、细胞间相互作用和表观遗传重编程如何在人类原肠胚形成过程中原始生殖细胞（PGC）（卵子或精子的前体）的规范中发挥作用。虽然已经在小鼠身上详细研究了PGCs的出现，但由于无法获得人类胚胎，因此不可能进行类似的研究。因此，人类发育的2D/3D体外胃原体模型将用于解决以下问题：产生PGCs的祖细胞的身份，形态梯度和表观遗传重塑的组合驱动PGC规范，以及定义的生态位在产生PGCs中的作用。总的来说，这些空间分辨单细胞方法的发展将为未来调制不同应用的细胞身份提供关键机会。该项目由分子和细胞生物科学部的系统和合成生物学集群支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Complex biological organisms arise from a single cell. Yet our understanding of how this single cell divides and differentiates into all of the specialized cells that make up different tissues within an organism is quite limited. The goal of this proposal is to understand how signaling cues arising from the spatial location of a cell within a tissue and its epigenetic state regulate gene expression during early human development. The function of a cell is tightly controlled by the cells location within a tissue and its intrinsic epigenetic landscape. While single-cell sequencing has revolutionized our understanding of mammalian systems by measuring the transcriptome and epigenome landscape within single cells, spatial information is lost using current methods. To better understand how spatial organization regulates gene expression in individual cells, the PI proposes to use photo-sensitive oligonucleotides to optically stamp the spatial positions of cells during development. This spatial information will be combined with single-cell mRNA sequencing and single-cell epigenetic features. Collectively, these techniques will map how spatial and epigenetic determinants control the specification of human primordial germ cells during early development. The data generated by the research aims will be used for a multi-layered and integrated educational program that includes a computational genomics bootcamp for high school students, a summer wet lab internship for undergraduates, and a mobile app game that introduces gene regulation concepts and targets high school students.To comprehensively study how spatial organization regulates gene expression of individual cells, the PI will design cholesterol-tagged photo-sensitive oligonucleotides (PSO) that incorporate into cell membranes, enabling an optical ‘stamp’ of the position of each cell prior to tissue dissociation and single-cell mRNA sequencing. Quantifying both mRNA and the relative degradation of the PSOs in individual cells will enable mapping spatial single-cell transcriptomes at high-resolution. Further, the PI will develop spatially-resolved single-cell multiomics technologies to simultaneously sequence the transcriptome together with different epigenetic features from the same cell to directly relate how DNA methylation, DNA accessibility and histone marks tune gene expression in varied spatial contexts. Furthermore, by employing iterative rounds of optical labeling, they will capture cell migration with end-point single-cell multiomics sequencing to gain insights into how tissue morphogenesis impacts cellular phenotypes. These transformative methods will be used to specifically study how morphogen gradients, cell-cell interactions, and epigenetic reprogramming play a role in the specification of primordial germ cells (PGC), the precursors to egg or sperm, during human gastrulation. While the emergence of PGCs has been studied in detail in mice, lack of access to human embryos makes similar studies impossible. Therefore, 2D/3D in vitro gastruloid models of human development will be used to address the following: What is the identity of the progenitors that give rise to PGCs, what combination of morphogen gradients and epigenetic remodeling drive PGC specification, and the role of defined niches in giving rise to PGCs. Overall, the development of these spatially-resolved single-cell methods will provide key opportunities in the future to modulate cell identity for varied applications. This project is supported by the Systems and Synthetic Biology Cluster of the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.