Live-imaging of endothelial Erk activity reveals dynamic and sequential signalling events during regenerative angiogenesis

Abstract

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 developing epicardium regulates cardiac chamber morphogenesis by promoting cardiomyocyte growth

“…To test this hypothesis, we first assessed the activation of the mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (ERK) pathway, a downstream effector of both FGF and VEGF signaling, in CMs. To do so, we generated a transgenic line, Tg(myl7:ERK-KTR-Clover-P2A-H2B-mScarlet) , to monitor ERK activation through a kinase translocation reporter (de la Cova et al, 2017; Mayr et al, 2018; Okuda et al, 2020) (Fig. 4D).…”

In vivo imaging of calcium dynamics in zebrafish hepatocytes

“…In the future, it would be interesting to investigate the regenerative pathways activated by the cell‐death–induced calcium spike. Particularly, it has recently been shown that the extracellular signal‐regulated kinase (ERK) pathway is activated by calcium signaling in a regenerative model of vascular damage in zebrafish 47…”

A kinase translocation reporter reveals real-time dynamics of ERK activity in Drosophila

“…Here we report the adaptation of the KTR technology for capturing ERK activity dynamics in Drosophila. Although initially developed for use in mammalian cell culture (Regot et al, 2014), ERK-KTR has since been successfully adapted in C. elegans , zebrafish and mouse and has enabled visualisation of ERK kinetics in a longitudinal manner (de la Cova et al, 2017; Mayr et al, 2018; Okuda et al, 2021; Pokrass et al, 2020; Simon et al, 2020). Combining the powerful advantages of Drosophila genetics with tissue-specific expression using the Gal4/UAS or Q systems means that Drosophila ERK-KTR is a versatile tool that will enable precise dissection of the dynamics of ERK signalling in a variety of physiological contexts.…”