Transcription factors networks in formative pluripotency

The transition from one cell state into the next is a hallmark of embryonic development. Each transition is accompanied by changes in gene expression profiles: The genes specific for one state must be turned off whereas the genes important for the next state must be activated. When which gene is activated is controlled by a network of transcription factors, that again is specific for each state. The transcription factor network that controls the mouse embryonic stem cell state has been characterized in depths and we understand most interactions among the core regulatory factors. However, once differentiation of the embryonic stem cells into formative pluripotency is initiated, a new and so far not well understood regulatory network is installed. We have identified previously Otx2 as one factor that is involved in the establishment of formative pluripotency. However, loss of Otx2 has only minor effects on the overall gene expression landscape, and cells are able to differentiate without major problems. This suggests that other, so far unknown factors are redundant with Otx2. To identify such factors, we will carry out a genome wide CRISPR-Cas9 based screen. We will take advantage of fluorescent markers that are reporting on the change in cell state. Furthermore, we will perform the screen in wild type cells and in cells that are deficient for Otx2. We hypothesize that the latter are more sensitive to loss of factors that show redundancy to Otx2. We will validate the candidates and analyze how they might genetically interact with Otx2. In the second part of the project we will analyze how loss of a single factor impacts differentiation on the single cell level. Loss of Otx2 does not impair the overall establishment of formative pluripotency. But do cells differentiate in exactly the same way as wild type cells? Do they follow the exact same trajectory or can we identify a potential novel state that cells deficient for a factor enter that we cannot identify in the wild type differentiation or through population wide measures? To address this question, we will combine a novel multiplexing strategy for single cell RNA-Seq that allows us to analyze multiple cell types and time points in one experiment. Together with recent advances in scRNA data analysis we will disentangle the exit from nave pluripotency on the single cell level.

During development, cells undergo numerous cell state transitions. With each transition, differentiating cells change their gene expression profile in reaction to a stimulus and assume, thereby, a new cellular identity. A network of transcription factors safeguards each developmental stage, but with each transition, the transcription factor network has to be required; some factors are inactivated, others are newly integrated into the system and thereby, gene expression changes are initiated. Each cell state is characterised by a specific network of transcription factors, and understanding the nature of such a network is one of the core question in developmental biology and transcriptional regulation. With the funded work, we set out to identify the key players of one cellular state: the formative pluripotent stem cell state. Murine embryonic stem cells, often referred to as nave pluripotent stem cells, are one of the best-characterized cellular states, and their transcription factor network has been studied in depth; however, as soon as the cells initiate differentiation, the cells exit the nave pluripotent state and enter the formative pluripotent state. While the formative pluripotent state shares many of the key transcription factors with the nave state, many of the nave specific transcription factors are downregulated and potentially replaced by other, unknown factors. However, the nature of these factors and their interplay with common transcription factors is unclear. Therefore, studying the formative transcription factor network will help us understand how cells transition from one cell state to another, which is a closely related cell state. In this project, we performed a genomic screen to identify important transcription factors for the formative pluripotent state. While we did not yet identify novel key players, we were able to characterise a new and unexpected antiviral response of the formative state that is activated during the transition from the nave to the formative state. This activation does not occur through the canonical signaling pathways monitoring viral infection but occurs through the pluripotency network itself. We propose, therefore, a novel model in which the developing embryo protects itself from viral infection through preemptive activation of an antiviral state. This study identified numerous additional candidates that could be important for the entrance in formative pluripotency and are currently under further investigation. In addition, our data contributed to developing a novel software tool that can predict changes in the transcription factor network upon loss of key components.