DEVELOPMENT OF SINGLE-CELL MASS SPECTROMETRY TOOLS TO INVESTIGATE METABOLIC REORGANIZATION DURING EARLY EMBRYOGENESIS
Portero, Erika Paola
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Measurement of metabolism in single cells holds the potential to advance our understanding of fundamental biological processes during cell differentiation and development. However, to characterize the metabolic state of single cells, further technological advances are still required. This dissertation discusses the development and application of single-cell mass spectrometry (MS) technologies to investigate metabolism and its role during tissue induction in the early developing vertebrate (frog) embryo. The work presented herein illustrates the strategies devised to advance single-cell analysis using capillary electrophoresis (CE)-MS. Additionally, this work features several contributions to our understanding of cell heterogeneity and the role of small molecules during tissue specification in the vertebrate embryo, providing new information to advance cell and developmental biology.Chapter 1 overviews the current state of metabolomics for cell and developmental biology, as well as the research significance and motivations. Chapter 2 describes the fundamental concepts of CE and the current state of single-cell metabolomics by CE-MS. This chapter also discusses the development of a minimally invasive microprobe sampling technique designed for the Xenopus laevis embryo. Chapter 3 presents the development of a CE-MS approach that enables dual cationic and anionic analysis of metabolites from the same single embryonic cell to deepen the detectable coverage of metabolism. Chapter 4 discusses a stable-isotope labeling strategy and single-cell CE-MS to uncover metabolic pathways involved in cell differentiation. Chapter 5 details the application of our custom-built microprobe sampling technique to investigate spatial cell heterogeneity in the same vertebrate embryo. This chapter examines cell-to-cell communication and small molecule transport between adjacent cells. Moreover, dual-fluorescent cell lineage tracing reveals cell fate changes induced by small molecule transport. Chapter 6 summarizes the results generated from this dissertation work and reflects on technical challenges and potential advancements needed to drive the field of MS-based single-cell metabolomics forward.