Quantitative Landscape of Xenopus laevis Transcriptome Dynamics During Lineage Restriction

Johnson, Kristin Lorette

doi:https://doi.org/10.21985/n2-ndn3-7742

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Quantitative Landscape of Xenopus laevis Transcriptome Dynamics During Lineage Restriction

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One of the fundamental questions in developmental biology is how a single cell gives rise to a complex organism. More specifically, how a totipotent egg divides into cells that become increasingly restricted in their potential. Development is a process of increasingly restricted cellular potential, and here I home in on the transition from pluripotent cells that are able to give rise to all cell types, to multipotent germ layers with differing and finite progenitor populations. I examine this transition from pluripotency to multipotency at high resolution at the transcriptome level in order to understand the dynamics of the transcriptome as cells exit pluripotency. Important biology is missed in our understanding of the exit from pluripotency when only the start and end points are studied. Much of the transition out of pluripotency and toward specific multipotent cell populations is driven by genes transiently expressed in a non-monotonic fashion, and thus a high resolution study is needed to understand how cells are able to exit pluripotency and how the transcriptome changes differ in cells pushed towards different germ layers.In this thesis, I developed a highly reproducible pipeline for analyzing the Xenopus transcriptome at six time points in the transition from pluripotency to four multipotent cell populations, endoderm, mesoderm, and the ectodermal derivatives, epidermis and neural progenitor cells. I provide quantitative support for the neural default model by demonstrating that the path to the neural lineage is the shortest and most linear, as these cells achieve an early equilibrium in their transcriptome dynamics not seen in the other three lineages. I identify novel divergent roles for BMP signaling before and after developmental stage 10.5. It has long been known that BMP signaling, in part, drives epidermal formation. Here I show that the epidermal and neural lineages have a largely shared trajectory until stage 10.5, at which point BMP signaling is activated in the epidermal lineage only, and these two lineages diverge. Importantly, early activation of BMP signaling does not drive an early divergence between these two lineages, but rather pushes cells to a mesodermal fate, suggesting cells are not able to appropriately respond to BMP to form epidermis until the time it comes endogenously, stage 10.5. The mechanism by which BMP signaling is held off until the appropriate time is driven, in part, by the presence of the maternally deposited transcription factor, Dand5. I also show that exposure to high levels of Activin leads to an early divergence in the transcriptome that is unique to endoderm cells, and I use this high resolution transcriptome data to provide temporal resolution to a previously established gene regulatory network (GRN) for the mesendoderm, as well as predict novel members of this GRN. The work in this thesis enhances our understanding of how cells progress from pluripotent to multipotent cell populations and provides valuable insight into the transcriptome at several time points during this transition to four lineages, providing a framework by which to identify previously identified key players in this transition, and to establish novel gene regulatory networks and enhance already established networks.

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