PgmNr Z599: Calcium signalling mediated by tmem33 is essential for endothelial tip cell function during angiogenesis in zebrafish.

Authors:
A. M. Savage 1,2 ; H. R. Kim 2 ; E. Markham 2 ; E. Honore 3 ; F. J. M. van Eeden 2 ; T. J. A. Chico 1,2 ; R. N. Wilkinson 1,2


Institutes
1) Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Western bank, Sheffield, UK; 2) Bateson Centre, Firth Court, University of Sheffield, Western bank, Sheffield, UK; 3) Institut de Pharmacologie Moleculaire et Cellulaire, Nice, France.


Abstract:

During angiogenesis, endothelial cells (ECs) sprout from pre-existing vessels driven by the response of endothelial tip cells to a gradient of VEGF. Stalk cells, which are determined through lateral inhibition via Notch signalling, are unable to respond to VEGF signals and migrate under the guidance of the tip cell. Calcium signalling is activated in ECs following stimulation by VEGF, which is dependent upon function of the calcium release-activated calcium (CRAC) channel. We have generated a transgenic zebrafish reporter of calcium signalling in ECs (fli1-gff; uas-gcamp7a). Using light sheet microscopy, we are able to visualise real-time EC calcium signalling in vivo. Inhibition of TRPV4 reduces calcium oscillations and delays segmental artery (SeA) migration, indicating TRPV4 may play a role in SeA development.

TMEM33 is a 3-transmembrane domain protein and putative component of the calcium signalling pathway which interacts with known calcium channels, PKD1 and PKD2 in mice. Zebrafish tmem33 morphants exhibit angiogenic branching defects. Furthermore, tmem33 knockdown attenuates EC calcium signalling, decreases EC filopodial dynamics and delays segmental artery migration, suggesting morphant ECs display a reduced response to VEGF signalling. Overexpression of VEGF mRNA does not rescue the tmem33 morphant phenotype. Inhibition of VEGF in zebrafish ECs also attenuates calcium signalling. Collectively, these data indicate tmem33 lies downstream of VEGF. Conversely, inhibition of Notch signalling in zebrafish embryos increases the frequency of calcium oscillations in ECs during angiogenesis. Zebrafish mindbomb mutants, which are Notch signalling deficient, exhibit increased tmem33 expression, while tmem33 morphants display attenuated Notch signalling, indicating Notch may be involved in negative feedback regulation of tmem33.

Interestingly, tmem33 mutants display nonsense-mediated decay of tmem33 transcripts, yet exhibit normal angiogenesis. tmem33 mutants also exhibit resistance to tmem33 morpholinos, suggesting genetic compensation exists in the tmem33 mutant.



ZFIN Genetics Index
1. tmem33
2. trpv4