Understanding Gene Regulation In Development And Differentiation Using Single Cell Multi-Omics
Transcriptional regulation is a major determinant of tissue-specific gene expression during development. My thesis research leverages powerful single-cell approaches to address this fundamental question in two developmental systems, C. elegans embryogenesis and mouse embryonic hematopoiesis. I have also developed much-needed computational algorithms for single-cell data analysis and exploration. C. elegans is an animal with few cells, but a striking diversity of cell types. In this thesis, I characterize the molecular basis for their specification by analyzing the transcriptomes of 86,024 single embryonic cells. I identified 502 terminal and pre-terminal cell types, mapping most single cell transcriptomes to their exact position in C. elegans’ invariant lineage. Using these annotations, I find that: 1) the correlation between a cell’s lineage and its transcriptome increases from mid to late gastrulation, then falls dramatically as cells in the nervous system and pharynx adopt their terminal fates; 2) multilineage priming contributes to the differentiation of sister cells at dozens of lineage branches; and 3) most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state. Next, I studied the development of hematopoietic stem cells (HSCs). All HSCs come from a specialized type of endothelial cells in the major arteries of the embryo called hemogenic endothelium (HE). To examine the cellular and molecular transitions underlying the formation of HSCs, we profiled nearly 40,000 rare single cells from the caudal arteries of embryonic day 9.5 (E9.5) to E11.5 mouse embryos using single-cell RNA-Seq and single-cell ATAC-Seq. I identified a continuous developmental trajectory from endothelial cells to early precursors of HSCs, and several critical transitional cell types during this process. The intermediate stage most proximal to HE, which we termed pre-HE, is characterized by increased accessibility of chromatin enriched for SOX, FOX, GATA, and SMAD binding motifs. I also identified a developmental bottleneck separates pre-HE from HE, and RUNX1 dosage regulates the efficiency of the pre-HE to HE transition. A distal enhancer of Runx1 shows high accessibility in pre-HE cells at the bottleneck, but loses accessibility thereafter. Once cells pass the bottleneck, they follow distinct developmental trajectories leading to an initial wave of lympho-myeloid-biased progenitors, followed by precursors of HSCs. During the course of both projects, I have developed novel computational methods for analyzing single-cell multi-omics data, including VERSE, PIVOT and VisCello. Together, these tools constitute a comprehensive single cell data analysis suite that facilitates the discovery of novel biological mechanisms.