Flk-1 (Foetal liver kinase-1), a receptor tyrosine kinase, and its ligand VEGF (vascular endothelial growth factor) are essential for vasculogenesis and haemopoiesis in the early embryo. Flk-1 is expressed initially on the haemangioblast, the common precursor of haemopoietic and endothelial cells, and on subsequent committed endothelial lineages. Flk-1 expression is maintained on adult endothelial cells and mediates angiogenesis, making it an important pharmaceutical target in the pathology of many human diseases including cancer and rheumatoid arthritis. Though involvement of Flk-1 in disease has lead to its extensive study in adult humans little is known about the signals it mediates during development.
Using ES cells as a model of development, the aim of this investigation was to identify signals mediated through Flk-1 during early development and to characterise their relative importance in the formation of the haemopoietic, endothelial and cardiomyocyte lineages. Activation of the MAPK and PLC signalling pathways were demonstrated following VEGF treatment of Flk-1-expressing embryoid bodyderived cells, though surprisingly activation of Flk-1 did not appear to mediate activation of the PI3K signalling pathway. Use of an embryoid body-based endothelial sprouting assay demonstrated a requirement for Flk-1 in both endothelial specification and angiogenic expansion. However, this activity was not mediated through either the MAPK or PI3K pathways. The finding that the PI3K pathway is not activated following VEGF stimulation nor required for early vasculogenesis/angiogenesis is surprising given its important role in both homeostatic and pathological angiogenesis in the adult. Previous work had suggested a role for Flk-1 in cardiac differentiation. Investigation of cardiomyocyte differentiation using Flk-1 null ES cells demonstrated a delay in formation of beating cardiomyocytes suggesting that Flk-1 may be involved in, but not required for cardiomyocyte specification. Finally, Flk-1-Tet-on ES cell lines were generated to facilitate investigation of the temporal importance of Flk-1 signalling during different developmental processes. Due to unforeseen difficulties in the maintenance of Flk-1 expression upon ES cell differentiation the full potential of this system was not realised in this study.
|Date of Award||1 Sept 2007|
|Supervisor||Melanie Welham (Supervisor)|