Cerebrovascular endothelial cells form transient Notch‐dependent cystic structures in zebrafish

Abstract

We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures “kugeln”, after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch‐dependent NO‐containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

Endothelial Cell Dynamics in Vascular Development: Insights From Live-Imaging in Zebrafish

“…Visualize F-actin polymerization on F-actin-based structures in endothelial cells such as filopodia and endothelial cell junctions Helker et al, 2013;Phng et al, 2013;Sauteur et al, 2014;Aydogan et al, 2015;Gebala et al, 2016;Hamm et al, 2016;Sauteur et al, 2017;Sugden et al, 2017;Paatero et al, 2018;Weijts et al, 2018;Kugler et al, 2019;Savage et al, 2019 Tg(4XUAS:mClavGR2-Has.UTRN) ubs27…”

“…Intersegmental vessels that remain arterial have a multicellular intersegmental vessel base while intersegmental vessels that will become venous intersegmental vessel have a unicellular base. Herwig et al, 2011;Lenard et al, 2013;Aydogan et al, 2015;Ando et al, 2016;Paatero et al, 2018;Geudens et al, 2019 (Continued) Frontiers in Physiology | www.frontiersin.org Chi et al, 2008;Fujita et al, 2011;Phng et al, 2013;Gebala et al, 2016;Eisa-Beygi et al, 2018;Kugler et al, 2019 Endothelial cell apical membrane reporter…”

“…The cerebral vessels in zebrafish develop under the control of brain specific molecular pathways (reviewed in Hogan and Schulte-Merker, 2017). Imaging of developing cerebral vessels revealed the formation of striking, large, transient, vesicle-like membranous protrusions, termed kugeln, that never detach from the parental ECs (Kugler et al, 2019). Kugeln do not have an EC nucleus and contain little cytoplasm, but dynamic F-actin polymerization is observed at the kugeln neck.…”

“…Kugeln do not have an EC nucleus and contain little cytoplasm, but dynamic F-actin polymerization is observed at the kugeln neck. Similar to cellular blebs, inhibition of F-actin polymerization using Latrunculin B increases kugeln number, while Myosin II inhibition by Blebbistatin reduces kugeln number (Charras et al, 2008;Kugler et al, 2019). Kugeln formation is modulated by various signaling pathways such as Vegfa/Kdr/Kdrl, Notch, and Wnt signaling, but their formation is not dependent on blood flow (Kugler et al, 2019).…”

The effect of absent blood flow on the zebrafish cerebral and trunk vasculature

“…Cerebral EC dysfunction is associated with neurodegenerative diseases, arteriovenous malformations, aneurysms, and stroke (Feigin Valery L. et al, 2017;Lackland and Weber, 2015). Increasing evidence suggests ECs display different molecular and functional properties according to their anatomical site such as the cerebral or trunk vessels (Abbott et al, 2010;Huntley et al, 2014;Kugler et al, 2019b;Vanlandewijck et al, 2018). This suggests novel insights could be obtained by studying the differential responses of these vascular beds to experimental manipulations.…”