Role of HYPPO/YAP Mechanosignalling in early development of the haematopoietic system

The first working package (WP) would be the generation of fluorescent reporter cell lines to visualize endothelial-to-haematopoietic transition (EHT) and involvement of the Hippo Pathway signalling in this process. EHT marks the crucial first step of successful development of an adult blood system and takes place in the embryo. Fluorescent proteins (like green- or yellow fluorescent protein [GFP, YFP]) emit photons of specific wavelength upon excitation. Furthermore, they can be genetically coupled to a protein of interest and then added to the genome of cells. This enables us to engineer reporter cell lines that allow optical imaging of both expression and intracellular localization of a protein of our choice. The Hippo effector protein YAP exhibits different functions, depending on intracellular localization (cytosolic or nuclear). By constructing a fluorescent YAP-reporter cell line, we could differentiate between the two modes of action with high-contrast and temporal resolution. Additionally, by coupling other proteins of interests (say haematopoietic differentiation marker RUNX1) to fluorescent proteins with different emission/excitation-spectra, we would be able to image the proteins of interest in parallel and could gain insight on Hippo/YAP contribution on haematopoietic differentiation in human embryonic stem cell (hES).
The second WP would be the genetical alteration of YAP expression and consequences thereof on development and differentiation of hES. This could be done by measures of CRISPR/Cas9. By altering gene expression in the reporter cell line from the previous working package, we hope to gain insight into the causal relationship between Hippo/YAP and EHT.
The third WP concerns the mechanosensory property of YAP and its importance in the context of EHT. YAP has been shown in vitro to switch between previously mentioned modes depending on physical properties (e.g. stiffness) of the surrounding cellular microenvironment.
By culturing the previously mentioned fluorescent cell line on substrate with modified physical properties, we may produce insight on how the microenvironment leads to Hippo/YAP-mediated switching of specific genes that could be responsible for differentiation of haematopoietic stem cells (HSCs). Additionally, by analysing the physical properties of specific embryonic tissue, which is enriched during EHT, we might produce substantial insight, that could help in formulating a comprehensive description of the microenvironment in vivo that could lead to successful generation of HSCs in vitro.