Metabolic characterization of single embryonic cells offers a direct snapshot of molecular events, raising the potential to understand biochemical processes that underlie healthy or pathological status during embryogenesis. To enable single-cell metabolic characterization for cell and developmental biology, several technological developments are needed. In this dissertation, we lay down the foundation for highly selective and sensitive analytical advances based on mass spectrometry (MS), that are compatible with the limited material afforded by single cells, and hence enable unbiased quantitation of diverse types of small biomolecules in the cells.
The work presented herein shows the outcome of my efforts to develop strategies and methodologies based on capillary electrophoresis-MS to investigate chemical heterogeneity between single embryonic cells during early stages of embryonic development. Additionally, the work features my contributions to the fundamentals of cell biology, and how metabolite activity is involved during early embryogenesis.
Chapter 1 discusses the basic biological and technological motivations for this dissertation.
Chapter 2 introduces current mass spectrometry-based techniques, including chemical separation by capillary electrophoresis, for the metabolic characterization of single cells.
Chapter 3 presents our development of single-cell capillary electrophoresis mass spectrometry for embryos. This chapter also discusses our discovery of metabolite-induced cell fate changes in the embryo of the vertebrate frog Xenopus laevis.
Chapter 4 details a multi-solvent extraction strategy to identify metabolites deeper into the single-cell metabolome. Additionally, we utilize this approach to uncover previously unknown metabolic cell heterogeneity between cells that occupy the left vs. right sides of the early developing X. laevis embryo.
Chapter 5 is dedicated to the development of a microprobe strategy to measure cell metabolism in situ in the live embryo. We demonstrate, for the first time, single-cell metabolomics in both the spatial and temporal dimension of embryonic development.
Chapter 6 injects the concept of spatially resolved metabolomics of multiple cells in the same freely developing embryo, focusing on the left-right developmental axis.
Chapter 7 reflects on the current state of metabolic single-cell mass spectrometry, spanning from technical challenges to new investigative potential in basic and translational research.
|Commitee:||Dowd, Cynthia S., Vertes, Akos|
|School:||The George Washington University|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 79/05(E), Dissertation Abstracts International|
|Subjects:||Analytical chemistry, Biochemistry, Developmental biology|
|Keywords:||Capillary electrophoresis, Embryo development, Mass spectrometry, Metabolomics, Single cell analysis, Xenopus laevis|
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