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

University Of California-Santa Barbara, Santa Barbara CA

Investigators

Abstract

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.