Uncovering regulators of cellular differentiation using single-cell sequencing

Abstract

New technologies have paved the way for addressing previously unanswerable questions. With rapid advancements in single-cell sequencing techniques, it is now possible to measure tens of thousands of molecules in millions of cells, revolutionizing the study of differentiation and fate choice. While these measurements provide a high-dimensional description of cellular differentiation, they do not directly identify the factors that regulate differentiation or the mechanisms by which they accomplish it. To tackle this challenge, it is essential to perturb processes that may be crucial for cell fate and then measure their effect on cellular differentiation. My PhD research is centered on this theme, with a particular focus on elucidating the roles of cell cycle and signaling in differentiation. Cell cycle and differentiation in embryonic development: Cell cycle and cellular differentiation are coupled throughout development. As cells differentiate, the rate of cell division changes, and the principal cell cycle regulators also vary across development. While previous studies have shown that embryos can still undergo widespread morphogenesis and survive for several days under cell cycle arrest, they exhibit many patterning defects. Thus the question of the role of cell division in the formation of the full diversity of embryonic cell types remains unanswered. To address this, we investigated the effects of cell cycle arrest in developing zebrafish embryos by single-cell RNA sequencing and found that cell division is dispensable for the differentiation of all major cell types. However, after arrest, differentiation slows down non-uniformly across multiple cell types. In contrast to differentiation, the proportions of cells across cell types are not robust to division block, and these changes in proportions can partially be explained by differential proliferation of cell types. Our findings shed light on the role of cell division in differentiation and provide a framework for investigating the effects of perturbations in tens of tissues in embryos using single-cell RNA sequencing. Signaling and differentiation in airway epithelium: Receptor-mediated signaling plays a central role in tissue regeneration and is dysregulated in disease. We systematically analyzed the effect of 17 receptor-mediated signaling pathways on human airway epithelial cultures to identify changes in the abundance and phenotype of all epithelial cell types. This revealed convergent states induced by multiple ligands and diverse, ligand-specific responses in epithelial differentiation. We show that loss of canonical differentiation induced by multiple pathways is associated with cell cycle arrest, but that arrest is not sufficient to block differentiation. Using the signaling-response map, we show that a TGFB1-mediated response underlies specific aberrant cells found in multiple lung diseases, and we identify interferon responses in COVID-19 patient samples. This work offers a framework that enables systematic evaluation of tissue signaling responses.