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• W2951472622 abstract "Article18 June 2019free access Transparent process Cerebrovascular endothelial cells form transient Notch-dependent cystic structures in zebrafish Elisabeth C Kugler Corresponding Author [email protected] orcid.org/0000-0003-2536-6140 Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Max van Lessen WWU Münster, Faculty of Medicine, Institute for Cardiovascular Organogenesis and Regeneration, Münster, Germany Search for more papers by this author Stephan Daetwyler Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Department of Cell Biology, The University of Texas Southwestern, Texas, TX, USA Search for more papers by this author Karishma Chhabria Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Aaron M Savage Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Vishmi Silva Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Karen Plant Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Ryan B MacDonald Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Jan Huisken Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Morgridge Institute for Research, Madison, WI, USA Search for more papers by this author Robert N Wilkinson Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Stefan Schulte-Merker WWU Münster, Faculty of Medicine, Institute for Cardiovascular Organogenesis and Regeneration, Münster, Germany Search for more papers by this author Paul Armitage Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK Search for more papers by this author Timothy JA Chico Corresponding Author [email protected] orcid.org/0000-0002-7458-5481 Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Elisabeth C Kugler Corresponding Author [email protected] orcid.org/0000-0003-2536-6140 Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Max van Lessen WWU Münster, Faculty of Medicine, Institute for Cardiovascular Organogenesis and Regeneration, Münster, Germany Search for more papers by this author Stephan Daetwyler Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Department of Cell Biology, The University of Texas Southwestern, Texas, TX, USA Search for more papers by this author Karishma Chhabria Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Aaron M Savage Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Vishmi Silva Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Karen Plant Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Ryan B MacDonald Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Jan Huisken Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Morgridge Institute for Research, Madison, WI, USA Search for more papers by this author Robert N Wilkinson Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Stefan Schulte-Merker WWU Münster, Faculty of Medicine, Institute for Cardiovascular Organogenesis and Regeneration, Münster, Germany Search for more papers by this author Paul Armitage Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK Search for more papers by this author Timothy JA Chico Corresponding Author [email protected] orcid.org/0000-0002-7458-5481 Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK Search for more papers by this author Author Information Elisabeth C Kugler *,1,2, Max Lessen3, Stephan Daetwyler4,5, Karishma Chhabria1,2, Aaron M Savage1,2, Vishmi Silva1,2, Karen Plant1,2, Ryan B MacDonald1,2, Jan Huisken4,6, Robert N Wilkinson1,2, Stefan Schulte-Merker3, Paul Armitage1,‡ and Timothy JA Chico *,1,2,‡ 1Department of Infection, Immunity and Cardiovascular Disease, Medical School, University of Sheffield, Sheffield, UK 2The Bateson Centre, Firth Court, University of Sheffield, Sheffield, UK 3WWU Münster, Faculty of Medicine, Institute for Cardiovascular Organogenesis and Regeneration, Münster, Germany 4Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 5Department of Cell Biology, The University of Texas Southwestern, Texas, TX, USA 6Morgridge Institute for Research, Madison, WI, USA ‡These authors contributed equally to this work as senior authors *Corresponding author. Tel: +44 1142 2210 81; E-mail: [email protected] *Corresponding author. Tel: +44 1142 2223 96; E-mail: [email protected] EMBO Rep (2019)20:e47047https://doi.org/10.15252/embr.201847047 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis The endothelial cells of the cranial vasculature in zebrafish extrude transient spherical structures termed “kugeln” after the German for sphere. Kugeln only form on cerebral vessels and are highly reactive for nitric oxide but their function is currently unknown. Kugeln are a novel Notch-dependent NO-containing endothelial cell membrane structure restricted to the cerebral vessels in zebrafish, of currently unknown function. Kugeln do not communicate with the vessel lumen, form in the absence of flow and contain little or no cytoplasm. Inhibition of actin polymerisation, Myosin II, or Notch signalling reduce kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increase kugel formation. Introduction Formation of mature blood vessels requires a wide range of endothelial behaviours. These include proliferation, migration, anastomosis, lumen formation, remodelling and pruning, alongside recruitment of non-endothelial cell types such as pericytes and vascular smooth muscle cells 1-4. Many of these processes can be studied in vitro, but in vivo models of vascular development allow observation of endothelial behaviour within a multicellular and complex physiological environment. Because visualizing real-time embryonic vascular development in mammals is technically challenging, the zebrafish has become a widely applied model of vertebrate vascular development. Their translucency enables detailed observation of cellular behaviour without invasive instrumentation in vivo 5, 6. An increasing array of transgenic reporter lines that drive fluorescent gene expression in vascular cells is available. Coupling these with state-of-the-art imaging techniques, such as light sheet fluorescence microscopy (LSFM), enables detailed cellular and subcellular imaging for hours or even days during embryonic development 7, 8. This ability to observe vascular development in more detail and for longer durations provides new insights into blood vessel formation. Endothelial and other cells are known to form a variety of membranous vesicles 9. These include apoptotic bodies (1–4 μm diameter), microvesicles (0.15–1 μm diameter) and exosomes (40–150 nm diameter) 10. There is increasing evidence that such vesicles play important roles in intracellular signalling 11 and in vascular diseases such as atherosclerosis 12. Understanding the roles of such vesicles is therefore of both biological and clinical significance. Here, we report a novel type of endothelial cell vesicle formation with characteristics that are entirely distinct from any previously described membrane behaviour. We show that endothelial cells (ECs) of the zebrafish cerebral vasculature (but not other vessels) extrude spherical structures far larger than any previously described microvesicle 10, and unlike previously described vesicles never detach from the parent endothelial cell. Due to their unknown nature and function, we termed these structures kugeln (German for spheres) and we here characterize their morphology, dynamics, and sites of occurrence. We find that kugeln are membranous protrusions that do not contain either nuclei or cytoplasm. Although very different in size and behaviour to other vesicular structures, inhibition of the cytoskeletal components filamentous actin (F-actin) formation or Myosin II reduced kugeln formation, as previously shown for cellular blebs 13. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) 14, 15 or macrophages, nor do they serve as scavengers. Kugeln formation is not influenced by either increased membrane rigidity, nor osmotic pressure, which are known to impact platelet ballooning and cellular blebbing 13, 16. Kugel formation can proceed in the absence of blood flow. Furthermore, we show that central orchestrators of vascular development such as Notch, VEGF and Wnt signalling 17-20 all influence kugel formation. Together, our data suggest that kugeln represent a previously undescribed EC membrane behaviour restricted to the cerebral vessels. Their function remains unknown, but their existence emphasizes that our understanding of the intricate processes during vascular development is far from complete. Results Endothelial cells of zebrafish cerebral vessels frequently develop large extruding spherical structures We initially observed that the endothelial cells of the cerebral vessels of transgenic Tg(kdrl:HRAS-mCherry)s916 zebrafish expressing an endothelial membrane-tagged reporter protein 21 displayed rounded protrusions (Fig 1A). Although the appearance of these on single z-slices was similar to cross-sections of lumenized vessels, three-dimensional reconstruction showed them to be spherical abluminal protrusions (Movie EV1). Due to their shape and unknown nature, we termed these structures kugeln, after the German for sphere (singular kugel). The mean diameter of kugeln was 10.1 ± 0.5 μm (s.e.m.) at 3 dpf (Fig 1B) exceeding the size of previously described membrane-derived vesicles 10. Figure 1. Endothelial cells of the zebrafish embryonic cerebral vessels develop “kugeln”; large spherical membrane protrusions MIP of cerebral vessels of 3 dpf Tg(kdrl:HRAS-mCherry)s916 embryo (grey LUT; inverted). Higher magnification panel showing two kugeln (arrowheads) arising from the middle mesencephalic central artery (MMCtA). Diameter of kugeln at 3 dpf (mean ± s.e.m. 10.13 ± 0.49; n = 93 kugeln from 32 3 dpf embryos; three experimental repeats). Number of kugeln per embryo in 3, 4 and 5 dpf animals was not statistically significantly different (mean ± s.e.m. 3 dpf 5.47 ± 1.09, 4 dpf 7.47 ± 1.46, 5 dpf 7.09 ± 1.55; P = 0.8571; 3 dpf: 175 kugeln from 32 embryos; 4 dpf: 239 kugeln from 32 embryos, 5 dpf: 227 kugeln from 32 embryos; four experimental repeats; Kruskal–Wallis test). Kugeln (arrowheads) could be observed in 28 dpf animals. MIP of cerebral vessels of 4 dpf Tg(fli1aep:CAAX-eGFP) embryo (grey LUT; inverted). Higher magnification panel shows a kugel (arrowhead) protruding from the posterior mesencephalic central artery (PMCtA). Single Z-plane micrograph of cerebral vessels of a 3 dpf Tg(gata1:dsRed), injected with a Tol2-fli1a:myr-Cherry construct showing two kugeln (arrowheads) in the higher magnification panel protruding from the MMCtA. Locations and proportion of kugeln on cerebral vessels (colour coded by vessel; total n = 107 kugeln from 34 4 dpf embryos; three experimental repeats). AMCtA—anterior mesencephalic central artery, BCA—basal communicating artery, CaDI—caudal division of internal carotid artery, CMV—communicating vessel, CtA—central arteries, MMCtA—middle mesencephalic central artery, MtA—metencephalic artery, PCS—posterior communicating segment, PMBC—posterior midbrain channel, PMCtA—posterior mesencephalic central artery, PrA—prosencephalic artery. Location of kugeln by vessel and laterality (107 kugeln from 34 4 dpf embryos; three experimental repeats comparison of left vs right P = 0.3592; Mann–Whitney U-test). Download figure Download PowerPoint Mean number of kugeln per embryo was 5.55 ± 1.12 (s.e.m.) at 3 dpf and was not significantly different between 3 and 5 dpf (P = 0.8571; Fig 1C). We were able to observe kugeln in 28 dpf animals (Fig 1D), confirming that kugeln are present beyond embryonic stages. To exclude the possibility that kugeln arise as a strain-specific feature of the transgenic line Tg(kdrl:HRAS-mCherry)s916, we imaged a different transgenic Tg(fli1aep:CAAX-eGFP) 22, which utilizes the pan-endothelial promotor fli1a 8 and prenylation to target eGFP to the endothelial membrane. Again, kugeln were observed to arise from cerebral vessels (Fig 1E). We furthermore induced transient expression of the plasmid pTol2-fli1a:myr-mCherry 23, 24, which uses the same fli1a promotor, and utilizes myristoylation to label the EC membrane. This was performed in Tg(gata1:dsRed) transgenics to enable vessel localization. Again, kugeln were observed in these embryos (Fig 1F), demonstrating that kugel formation was independent of the transgenic construct used, the promotor driving its expression and the method of membrane tagging. Therefore, we concluded that kugeln represent a physiological behaviour of the EC membrane of the cranial vasculature. We mapped the location of kugeln on parent vessels to determine their distribution. > 90% of kugeln arose from the central vessels of the cerebral vasculature (Fig 1G) and none from the trunk vasculature (aorta, intersegmental vessels, dorsal longitudinal anastomotic vessel or caudal vein) at the examined time-points. The specific vessels giving rise to kugeln are shown in Fig 1H. Although individual kugeln were often unilateral (present left-side but not right-sided vessels, or vice versa), overall left/right distribution was not significantly different (Fig 1H). Kugeln are highly dynamic transient structures We next observed kugel behaviour over time. Time-lapse imaging revealed kugeln were both transient and highly dynamic (Fig 2A). Although we expected kugeln to form new vessels, interact with other ECs to anastomose or detach from parent vessels, kugeln always either regressed back into the parent vessel or persisted to the end of imaging without separation or anastomosis. Figure 2. Endothelial kugeln are transient and dynamically alter shape and size MIPs of a time-series light sheet acquisition shows three different kugeln (kugel 1—black arrowhead; kugel 2—grey arrowhead; kugel 3—unfilled arrowhead) protruding and retracting from parent vessels (2 min intervals; inverted LUT). Kugeln persisted for variable durations before regression into the parent vessel (43 kugeln from 9 4 dpf embryos; four experimental repeats). MIPs taken 20 min apart showing examples of different kugel behaviour including shape changes (asterisk), expansion (circle), retraction (triangle) or little change (pentagon). MIPs taken 1 h apart show that kugeln may be observed at one time-point (red arrowhead, 55:20:00) but not 1 h later in the same animal (56:20:00) and to develop on other vessels 1 h later (57:20:00) which again have regressed by 58:20:00. Kymographs generated by line-scanning across the diameter of a typical kugel shows that kugel diameter oscillated with a periodicity of minutes, while no such oscillations were observed in adjacent similarly sized cerebral vessels (all images grey LUT; inverted). Download figure Download PowerPoint Quantification of kugel lifespan showed that while some regressed after minutes, others persisted for hours (Fig 2B). Further examination showed that kugeln displayed dynamic alteration in shape and size, including shape changes, enlargement and retraction (Fig 2C; Movie EV2). This dynamic behaviour was observed not only in Tg(kdrl:HRAS-mCherry)s916 animals but also in Tg(fli1ep:CAAX-eGFP) (Fig EV1A and B) and Tg(fli1a:myr-mCherry), Tg(gata1:dsRed) (Fig EV1C). Click here to expand this figure. Figure EV1. Kugeln were observed in the transgenic Tg(fli1aep:eGFP-CAAX) MIP of cerebral vessels of 4 dpf Tg(fli1aep:eGFP-CAAX) embryo. Time-lapse acquisitions showed that kugeln were dynamic (black arrowhead) as in Tg(kdrl:HRAS-mCherry)s916, showing protrusion, shape changes, oscillation and retraction. Additional time-lapse acquisitions in the Tg(fli1a:myr-mCherry), Tg(gata1:dsRed) showed equally dynamics of kugeln (black arrowhead; images grey LUT; inverted). Download figure Download PowerPoint Although some animals displayed no kugeln when observed at a single time-point (Fig 1C), time-lapse imaging showed kugeln could develop at subsequent time-points (Fig 2D). When we studied kugel shape change over time, this revealed that some kugeln displayed oscillatory diameter changes with a periodicity of minutes (Fig 2E). No such changes were observed in parent vessels, suggesting alterations of kugel size or shape are not directly related to blood pressure or flow but related to active membrane remodelling. Kugeln are membranous structures whose formation is dependent on the cytoskeleton We next investigated the composition and biogenesis of kugeln. Examining double-transgenic embryos Tg(kdr:nls-eGFP)zf109, Tg(kdrl:HRAS-mCherry)s916 that labelled endothelial nuclei and membrane, we never observed kugeln to contain a nucleus, nuclear defragmentation or observed nuclear mitosis nearby kugeln (Fig 3A). This suggests kugeln do not represent an atypical form of cell proliferation, apoptosis or angiogenic sprouting. Figure 3. Endothelial kugeln are non-nucleated with a filamentous actin-enriched neck Double transgenic visualizing endothelial membrane (red) and endothelial nuclei (green). Endothelial nuclei (arrowhead) were observed close to but never within the kugel (unfilled arrowhead). Double transgenic showing endothelial membrane (red) and endothelial F-actin (green). F-actin was found to localize at the neck of the kugeln (arrowhead; see also: Movie EV3). Double transgenic showing endothelial membrane (red) and endothelial cytoplasm (green). The cytoplasmic reporter was visible in the parent vessel but not in the kugel (unfilled arrowhead). Triple transgenic showing endothelial membrane (red), neurons (green) and erythrocytes (red). Surrounding neurons were excluded from the volume of the kugel (unfilled arrowhead), and erythrocytes were not observed inside kugeln (arrowhead). Treatment with the inhibitor of actin polymerization Latrunculin B statistically significantly increased number of kugeln per embryo (100 nM 1 h; **P = 0.0041; control n = 21 embryos 8.05 ± 1.76 (mean ± s.e.m.); Latrunculin n = 21 embryos 17.19 ± 2.93 (mean ± s.e.m.); 4 dpf; three experimental repeats; Mann–Whitney U-test). Latrunculin B treatment statistically significantly reduced kugel diameter (*P = 0.0164; control n = 169 kugeln from 21 embryos 7.56 ± 2.25 (mean ± s.e.m.); Latrunculin n = 361 kugeln from 21 embryos 5.91 ± 1.78 (mean ± s.e.m.); 4 dpf; three experimental repeats; Student's t-test). Treatment with the Myosin II inhibitor Blebbistatin statistically significantly reduced number of kugeln per embryo (25 μM 1 h; ****P < 0.0001; control n = 22 embryos 3.77 ± 0.56 (mean ± s.e.m.), Blebbistatin n = 24 embryos 1.08 ± 0.22 (mean ± s.e.m.); 3 dpf; three experimental repeats; Mann–Whitney U-test). Blebbistatin treatment had no effect on kugel diameter (P = 0.3731; control n = 83 kugeln from 22 embryos 6.27 ± 0.72 (mean ± s.e.m.), Blebbistatin n = 26 kugeln from 24 embryos 7.02 ± 0.89 (mean ± s.e.m.); 3 dpf; three experimental repeats; Mann–Whitney U-test). Download figure Download PowerPoint Analysing the double-transgenic line Tg(fli1a:LifeAct-mClover)sh467, Tg(kdrl:HRAS-mCherry)s916 labelling filamentous actin (F-actin) and EC membrane, we observed that F-actin co-localized with kugeln, especially at the kugel “neck” (Fig 3B). Time-lapse microscopy revealed this enrichment of F-actin at the kugel neck to be highly dynamic (Movie EV3). To further examine the contents of kugeln, we imaged the double-transgenic Tg(fli1a:eGFP)y1, Tg(kdrl:HRAS-mCherry)s916 that labels the EC cytosol and membrane. We were unable to visualize cytosolic GFP within kugeln, indicating they contain little if any cytoplasm (Fig 3C). To further characterize the content inside and tissue surrounding kugeln, we examined the triple-transgenic Tg(gata1:dsRed), Tg(nbt:GCaMP3), Tg(kdrl:HRAS-mCherry)s916 that labels red blood cells (RBCs), developing neurons and EC membrane. RBCs were never observed within kugeln. Examination of neurons showed an exclusion of neural tissue (Fig 3D), confirming kugeln displace rather than include neural tissue. Kugel formation is reduced by F-actin or Myosin II inhibition Since F-actin was enriched at kugel necks, we examined whether this was necessary for growth and/or maintenance of kugeln. Thus, actin polymerization was inhibited by application of Latrunculin B. This led to a significant increase of number of kugeln per embryo (Fig 3E; P = 0.0041), but a significant decrease in kugel diameter (Fig 3F; P = 0.0164). The role of Myosin II was investigated by chemical inhibition by Blebbistatin treatment. This significantly reduced the number of kugeln per embryo (Fig 3G; P < 0.0001) but did not affect kugeln diameter (Fig 3H; P = 0.3731). The finding that F-actin inhibition increased, while Myosin II inhibition decreased, kugel number is consistent with the effect of these manipulations on cellular blebs 13, suggesting some shared mechanisms of kugel and bleb biogenesis. Blood flow is not required for kugel formation, maintenance, retraction or oscillation To study the impact of blood flow and blood pressure, we performed exsanguination by cardiac puncture to reduce blood pressure and flow to zero. Imaging the same animals pre- and post-exsanguination showed no effect on kugel size or shape (Fig 4A and B), suggesting neither flow nor pressure are needed to maintain kugeln once they have formed. To confirm this, cardiac contraction was temporarily stopped by high-dose Tricaine application and time-lapses acquired. Despite absent blood flow, kugeln still developed, changed shape and retracted (Fig 4C and D) as seen under normal blood flow conditions. Figure 4. The relationship between endothelial kugeln and blood flow MIP of the cerebral vessels of a 4 dpf embryo before exsanguination (grey LUT; inverted). MIP of the same embryo after exsanguination, which did not alter kugel size. Time-lapse imaging of an embryo with transiently halted cardiac contraction (using Tricaine). Despite absent blood flow kugeln still changed shape (grey arrowhead), retained shape (white arrowhead) or protruded and retracted (black arrowhead; time post cessation of flow is indicated on micrographs; 3 dpf; grey inverted LUT). Time-lapse of an embryo with transiently halted cardiac contraction as in (C) showed that kugel diameter still oscillates (time post cessation of flow). Dextran microangiography filled perfused vessels with dextran (arrowhead), while dextran was not observed to enter kugeln (unfilled arrowhead). Inhibition of cardiac contraction by tnnt2a morpholino (MO) knockdown statistically significantly reduced kugel number per embryo (****P < 0.0001; control n = 20 embryos 5.10 ± 1.47 (mean ± s.e.m.), tnnt2a MO = 18 embryos 0.06 ± 0.06 (mean ± s.e.m.); 3 dpf; three experimental repeats; Mann–Whitney U-test). Download figure Download PowerPoint We next performed microangiography with fluorescent dextran to investigate whether kugeln were perfused by blood or communicated with the lumen of the parent vessel. No entry of dextran into kugeln was observed (Fig 4E), suggesting no such communication existed, at least after kugeln were formed. Lastly, we prevented the development of heart contraction by morpholino (MO) knockdown of cardiac troponin 2a (tnnt2a) 25-27. This induced a significant decrease in number of kugeln per embryo (Fig 4F; P < 0.0001). Together, these data suggest that blood flow is not the driving mechanism of kugel formation, retraction or oscillation, but that vessels that have never experienced flow do not form kugeln. Altered membrane permeability or osmotic pressure does not affect kugel formation or diameter Since DMSO increases membrane permeability 28, we examined whether this had an impact on kugel formation. We incubated embryos for 24 h in 2.5% DMSO and found no significant difference in kugel number (Fig EV2A; P = 0.1596) or diameter (Fig EV2B; P = 0.3665). Click here to expand this figure. Figure EV2. Kugeln number or diameter is not altered by changes of membrane permeability or osmotic pressure The influence of membrane permeability increase was studied by application of DMSO; the number of kugeln was not statistically significantly changed (2.5% DMSO 24 h; P = 0.1596; control n = 25 embryos 4.00 ± 0.65 (mean ± s.e.m.), DMSO n = 25 embryos 3.00 ± 0.62 (mean ± s.e.m.); 4 dpf; three experimental repeats; Mann–Whitney U-test). Diameter of kugeln was not statistically significantly different after incubation with DMSO (P = 0.3665; control n = 97 kugeln from 25 embryos 8.68 ± 0.71 (mean ± s.e.m.), DMSO n = 75 kugeln from 25 9.76 ± 0.67 (mean ± s.e.m.); 4 dpf; three experimental repeats; Student's t-test). The impact of osmotic pressure on kugeln was studied by application of glucose; no statistically significant difference was observed (40 mM glucose 24 h; P = 0.7371; control n = 22 embryos 2.46 ± 0.36 (mean ± s.e.m.), glucose n = 21 embryos 3.24 ± 0.65 (mean ± s.e.m.); 4 dpf; two experimental repeats; Mann–Whitney U-test). Kugel diameter was not statistically significantly different after incubation with glucose (P = 0.7060; control n = 54 kugeln from 22 embryos 10.09 ± 1.02 (mean ± s.e.m.), glucose n = 67 kugeln from 21 embryos 10.66 ± 4.65 (mean ± s.e.m.); 4 dpf; two experimental repeats; Student's t-test). Download figure Download PowerPoint As osmotic pressure increases cellular bleb formation and platelet ballooning 13, 16, we examined whether kugeln were altered by increased osmotic pressure. Embryos were incubated for 24 h in a 40 mM glucose solution. No significant difference was found in kugel number (Fig EV2C; P = 0.7371) or diameter (Fig EV2D; P" @default.
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• W2951472622 title "Cerebrovascular endothelial cells form transient Notch‐dependent cystic structures in zebrafish" @default.
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• W2951472622 doi "https://doi.org/10.15252/embr.201847047" @default.
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