The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation and migration. Erk is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response however, involves a Vegfr-dependent mechanism that initiates concomitant with the wound inflammatory response. This work reveals a highly dynamic sequence of signalling events in regenerative angiogenesis and validates a new resource for the study of vascular Erk-signalling in real-time.
The formation of new blood vessel networks occurs via angiogenesis during development, tissue repair and disease. Angiogenesis is regulated by intracellular endothelial signalling pathways, induced downstream of Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs). A major challenge in understanding angiogenesis is interpreting how signalling events occur dynamically within endothelial cell populations during sprouting, proliferation and migration. Erk is a central downstream effector of Vegf-signalling and reports the signalling that drives angiogenesis. We generated a vascular Erk biosensor transgenic line in zebrafish using a kinase translocation reporter that allows live-imaging of Erk-signalling dynamics. We demonstrate the utility of this line to live-image Erk activity during physiologically relevant angiogenic events. Further, we reveal dynamic and sequential endothelial cell Erk-signalling events following blood vessel wounding. Initial signalling is dependent upon Ca2+ in the earliest responding endothelial cells, but is independent of Vegfr-signalling and local inflammation. The sustained regenerative response however, involves a Vegfr-dependent mechanism that initiates concomitant with the wound inflammatory response. This work thus reveals a highly dynamic sequence in regenerative angiogenesis that was not previously appreciated. Altogether, this study demonstrates the utility of a unique biosensor strain for analysing dynamic endothelial Erk-signalling events and validates a new resource for the study of vascular signalling in real-time.
The assembly of a mature vascular network involves coordinated endothelial cell (EC) shape changes, including a process of EC elongation. How EC elongation is dynamically regulated in vivo is not fully understood. Here, we generated a zebrafish mutant, deficient for the integrin adaptor protein Talin1. Using a new Focal Adhesion (FA) marker line, expressing endothelial Vinculinb-eGFP, we demonstrated that EC FAs function dynamically and are lost in our talin1 mutants, allowing us to uncouple the primary roles of FAs in ECs morphogenesis, from the secondary effects that occur due to systemic vessel failure or loss of blood flow. Talin1 loss led to compromised F-actin rearrangements, perturbed EC elongation and disrupted cell-cell junction linearisation in vessel remodelling. Finally, chemical induction of actin polymerisation restored FA dynamics and EC elongation during vascular morphogenesis. Together, we have identified that FAs are essential for EC elongation and junction linearisation in flow pressured vessels and influence actin polymerisation in cellular morphogenesis. These observations can explain the severely compromised vessel beds and vascular leakage observed in mutant models that lack integrin signalling.
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