Blood Flow Changes Coincide with Cellular Rearrangements during Blood Vessel Pruning in Zebrafish Embryos Eva Kochhan 1 , Anna Lenard 2 , Elin Ellertsdottir 2 , Lukas Herwig 2 , Markus Affolter 2 , Heinz-Georg Belting 2 , Arndt F. Siekmann 1 * 1 Max Planck Institute for Molecular Biomedicine, Laboratory for Cardiovascular Patterning, Muenster, Germany, 2 Biozentrum der Universita ¨t Basel, Abteilung Zellbiologie, Basel, Switzerland Abstract After the initial formation of a highly branched vascular plexus, blood vessel pruning generates a hierarchically structured network with improved flow characteristics. We report here on the cellular events that occur during the pruning of a defined blood vessel in the eye of developing zebrafish embryos. Time-lapse imaging reveals that the connection of a new blood vessel sprout with a previously perfused multicellular endothelial tube leads to the formation of a branched, Y- shaped structure. Subsequently, endothelial cells in parts of the previously perfused branch rearrange from a multicellular into a unicellular tube, followed by blood vessel detachment. This process is accompanied by endothelial cell death. Finally, we show that differences in blood flow between neighboring vessels are important for the completion of the pruning process. Our data suggest that flow induced changes in tubular architecture ensure proper blood vessel pruning. Citation: Kochhan E, Lenard A, Ellertsdottir E, Herwig L, Affolter M, et al. (2013) Blood Flow Changes Coincide with Cellular Rearrangements during Blood Vessel Pruning in Zebrafish Embryos. PLoS ONE 8(10): e75060. doi:10.1371/journal.pone.0075060 Editor: Ben Hogan, University of Queensland, Australia Received July 5, 2013; Accepted August 8, 2013; Published October , 2013 Copyright: ß 2013 Kochhan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Max Planck Society (www.mpg.de; AFS), the Deutsche Forschungsgemeinschaft (DFG SI-1374/3-1; www.dfg.de; AFS) and an European Research Council starting grant (260794-ZebrafishAngio; erc.europa.eu; AFS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: arndt.siekmann@mpi-muenster.mpg.de Introduction The vasculature is the first organ system to form during embryonic development and meets the challenge to grow and refine while it is already functioning. After the initial sprouting of blood vessels, remodeling ensures the formation of a more efficient vascular network [1–3]. While many of the key factors and genetic players regulating blood vessel sprouting are known, the mecha- nisms which control blood vessel remodeling and pruning are only poorly understood. The best-studied context is the regression of the hyaloid vasculature in mice [4]. Here, macrophages secrete a WNT ligand that induces apoptosis in endothelial cells, ultimately leading to the complete removal of this vascular structure [5,6]. However, in many other contexts where vascular pruning has been described, such as the mouse retina [7,8], the branchial arches [9] or the zebrafish brain [10], only a subset of blood vessel connections is removed. This raises the question: Which mech- anism selects the blood vessels to be pruned? In addition, it is unclear how, once a given blood vessel is selected to disconnect and regress, the endothelial cells constituting this previously perfused vessel accomplish to seal it off from the active circulation without causing hemorrhage. Here, we use time-lapse imaging to analyze the cellular mechanisms that take place during the pruning of a defined blood vessel in the eye of zebrafish embryos. Our analysis reveals that angiogenic sprouting initially leads to the formation of a Y- shaped blood vessel branch, which is subsequently resolved. This process entails the rearrangement of endothelial cells within the pruning blood vessel from a multicellular to a partially unicellular tube. We show that blood flow is an important regulator of the pruning event. Importantly, our results suggest that loss of perfusion in itself does not lead to blood vessel pruning, but that pruning might be facilitated by the establishment of differences in blood flow between vessels in a branch point. Results and Discussion Live Imaging of Blood Vessel Pruning To better understand vascular remodeling, we set out to identify blood vessels that undergo pruning during zebrafish embryonic development. We performed time-lapse analysis of the eye vasculature between 32 hours post fertilization (hpf) and 48 hpf in Tg(kdrl:Hsa.HsRAS-mcherry) sa916 ; Tg(fli1a:nEGFP) y7 fish to label endothelial cell membranes and nuclei (Figure 1). These movies revealed that a discrete portion of the Cranial Division of the Internal Carotid Artery (CrDI) was pruned in a consistent manner (Figure 1, see also Movie S1). Initially, the Nasal Ciliary Artery (NCA) connected to the CrDI, dividing it into a dorsal and a ventral part (Figure 1C, arrow in D, naming according to [11]). After branch point establishment, we observed a collapse of endothelial lumen in the dorsal CrDI (Figure 1E, arrowhead), followed by blood vessel regression (Figure 1E, F, arrowheads). Imaging several zebrafish larvae furthermore showed that this pruning event occurred in a highly reproducible manner, PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e75060 11