Cutting edge tools to reveal lineages and epigenetic modifications in single cells

TY - THES

T1 - Cutting edge tools to reveal lineages and epigenetic modifications in single cells

AU - Florescu, Maria

PY - 2020/9/29

Y1 - 2020/9/29

N2 - Every cell in our body has a past, present and future that is coded in biomolecules. The history of a cell can be, for example, encrypted in small changes in the DNA. Once the DNA in a cell is changed, it transfers these changes to its future descendants. Studying a cell's past and present can help to make predictions about what this cell will be in the future. To go from a single cell to a complex organism of a billion cells, cells take action to specialize in different cell types. To follow these cellular differentiation processes it is crucial to measure multiple types of biomolecules. However, measuring DNA and RNA from the same single cell is still challenging. Single cells have only a small number of copies of these biomolecules and biomolecules are vulnerable to cell disruption, which is a necessary step to take measure cellular content. With sequencing technologies RNA or DNA in individual cells can be reliably read. In the first chapter, I provide an overview of the here presented technologies and their context in the single-cell sequencing field and related technologies. In the second chapter, we track a specific group of early stem cells in the zebrafish embryo. Similar stem cells in mice are known to proliferate in different cell types such as spinal cord and muscles. How these stem cells are behaving in the zebrafish is unclear and not well characterized. With CRISPR / Cas9 we are introducing a barcode at a very early embryonic stage in a large number of cells. We also track these specific cellular population of cells by microscopy in the young zebrafish embryo. Our results suggest that these stem cells indeed have a similar function as in the development of mice. In the third chapter, we present a new technology, ScarTrace, which combines the barcodes introduced by CRISPR / Cas9 with RNA sequencing. With these two types of single cell measurements, we can do both, determine the lineage of a cell as well as the cellt type. Our study shows that whole kidney marrow clones are found in all cell types we obtained from the whole kidney marrow, which supports the hypothesis that a few stem cell clones make all major blood cell types. Furthermore, our study suggests that cells that contribute to the left and right eye separated before zygotic gene activation. Finally, we identified a subpopulation of macrophages in the caudal fin which contribute to its tissue regeneration after injury. In the fourth chapter we introduce a new technology, scChIX, for reading two histone modifications within the same single cell. Epigenetic regulation is based on the interplay of different types of histone modifications to regulate the genome. We demonstrate that scChIX can deconvolve a mixed signal of two types of histone modifications in purified cell types from the bone marrow of a mouse. In the last chapter, I provide a discussion enhacing potential and pitfalls of the here presented technologies and their impact on cell identity.

AB - Every cell in our body has a past, present and future that is coded in biomolecules. The history of a cell can be, for example, encrypted in small changes in the DNA. Once the DNA in a cell is changed, it transfers these changes to its future descendants. Studying a cell's past and present can help to make predictions about what this cell will be in the future. To go from a single cell to a complex organism of a billion cells, cells take action to specialize in different cell types. To follow these cellular differentiation processes it is crucial to measure multiple types of biomolecules. However, measuring DNA and RNA from the same single cell is still challenging. Single cells have only a small number of copies of these biomolecules and biomolecules are vulnerable to cell disruption, which is a necessary step to take measure cellular content. With sequencing technologies RNA or DNA in individual cells can be reliably read. In the first chapter, I provide an overview of the here presented technologies and their context in the single-cell sequencing field and related technologies. In the second chapter, we track a specific group of early stem cells in the zebrafish embryo. Similar stem cells in mice are known to proliferate in different cell types such as spinal cord and muscles. How these stem cells are behaving in the zebrafish is unclear and not well characterized. With CRISPR / Cas9 we are introducing a barcode at a very early embryonic stage in a large number of cells. We also track these specific cellular population of cells by microscopy in the young zebrafish embryo. Our results suggest that these stem cells indeed have a similar function as in the development of mice. In the third chapter, we present a new technology, ScarTrace, which combines the barcodes introduced by CRISPR / Cas9 with RNA sequencing. With these two types of single cell measurements, we can do both, determine the lineage of a cell as well as the cellt type. Our study shows that whole kidney marrow clones are found in all cell types we obtained from the whole kidney marrow, which supports the hypothesis that a few stem cell clones make all major blood cell types. Furthermore, our study suggests that cells that contribute to the left and right eye separated before zygotic gene activation. Finally, we identified a subpopulation of macrophages in the caudal fin which contribute to its tissue regeneration after injury. In the fourth chapter we introduce a new technology, scChIX, for reading two histone modifications within the same single cell. Epigenetic regulation is based on the interplay of different types of histone modifications to regulate the genome. We demonstrate that scChIX can deconvolve a mixed signal of two types of histone modifications in purified cell types from the bone marrow of a mouse. In the last chapter, I provide a discussion enhacing potential and pitfalls of the here presented technologies and their impact on cell identity.

M3 - PhD thesis

PB - Utrecht University

ER -