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.
Zebrafish transgenic lines and light sheet fluorescence microscopy allow in-depth insights into vascular development in vivo and 3D. However, robust quantification of the zebrafish cerebral vasculature in 3D remains a challenge, and would be essential to describe the vascular architecture. Here, we report an image analysis pipeline that allows 3D quantification of the total or regional zebrafish brain vasculature. This is achieved by landmark- or object-based inter-sample registration and extraction of quantitative parameters including vascular volume, surface area, density, branching points, length, radius, and complexity. Application of our analysis pipeline to a range of sixteen genetic or pharmacological manipulations shows that our quantification approach is robust, allows extraction of biologically relevant information, and provides novel insights into vascular biology. To allow dissemination, the code for quantification, a graphical user interface, and workflow documentation are provided. Together, we present the first 3D quantification approach to assess the whole 3D cerebrovascular architecture in zebrafish.
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