© 2021. Published by The Company of Biologists Ltd | Development (2021) 148, dev194993. doi:10.1242/dev.194993 RESEARCH ARTICLE Vasohibin 1 selectively regulates secondary sprouting and lymphangiogenesis in the zebrafish trunk Marta Bastos de Oliveira1,2, Katja Meier1,2, Simone Jung1,2, Eireen Bartels-Klein1,2, Baptiste Coxam1,2, Ilse Geudens3,4, Anna Szymborska1,2, Renae Skoczylas3, Ines Fechner1,2, Katarzyna Koltowska3 and Holger Gerhardt1,2,4,5,6,* ABSTRACT The trunk vasculature in zebrafish embryos has served both Previous studies have shown that Vasohibin 1 (Vash1) is stimulated angiogenesis and lymphangiogenesis research as a powerful model by VEGFs in endothelial cells and that its overexpression interferes to observe, manipulate and mechanistically understand relevant with angiogenesis in vivo. Recently, Vash1 was found to mediate cellular processes and their molecular control (Gore et al., 2016; tubulin detyrosination, a post-translational modification that is Hogan and Schulte-Merker, 2017). Following the initial assembly implicated in many cell functions, such as cell division. Here, we and lumen formation of the dorsal aorta (DA) and posterior cardinal used the zebrafish embryo to investigate the cellular and subcellular vein (PCV), the intersegmental vessels (ISV) form by sprouting. mechanisms of Vash1 on endothelial microtubules during formation This first wave of sprouting emerges from the DA at 22 h post of the trunk vasculature. We show that microtubules within venous- fertilization (hpf), with bilateral sprouts along the somite derived secondary sprouts are strongly and selectively detyrosinated boundaries forming the first vascular loops (Isogai et al., 2003; in comparison with other endothelial cells, and that this difference is Kohli et al., 2013). A second wave of sprouting emerges at 34 hpf lost upon vash1 knockdown. Vash1 depletion in zebrafish specifically and consists of venous and lymphatic sprouts (Koltowska et al., affected secondary sprouting from the posterior cardinal vein, 2015; Nicenboim et al., 2015; Yaniv et al., 2006). The venous increasing endothelial cell divisions and cell number in the sprouts. sprouts remodel half of the ISVs into veins (Geudens et al., 2010; We show that altering secondary sprout numbers and structure upon Yaniv et al., 2006). The lymphatic precursor cells are marked by the Vash1 depletion leads to defective lymphatic vessel formation and transcription factor Prox1 in the PCV, before a cell division that ectopic lymphatic progenitor specification in the zebrafish trunk. results in a differential Prox1 distribution in the daughter cells, influencing their specification and behaviour. Cells retaining high KEY WORDS: Lymphangiogenesis, Vash1, Microtubules, Tubulin Prox1 levels contribute to sprouts that will form parachordal detyrosination, Sprouting angiogenesis lymphangioblasts (PLs) at the midline. The PLs subsequently migrate and branch to shape the mature lymphatic system of the INTRODUCTION zebrafish trunk including the thoracic duct (TD) and the dorsal Blood vessel formation and patterning is essential for tissue growth longitudinal lymphatic vessel (DLLV) (Küchler et al., 2006; Yaniv and homeostasis in vertebrate development and physiology. et al., 2006). Although primary and secondary sprouts appear Endothelial cells (EC) arising from the lateral plate mesoderm in morphologically similar (Isogai et al., 2003), with acto-myosin early embryonic development initially coalesce at the midline to cellular protrusions, they are driven by distinct growth factors and form the first arterial and venous structures. Subsequent sprouting downstream signalling cascades. In particular, arterial sprouting angiogenesis and remodelling expands and shapes the vascular tree involves Vegfa, Kdr and Plcγ, whereas venous/lymphatic sprouting throughout the developing embryo (Hogan and Schulte-Merker, is controlled by Vegfc, Flt4, Ccbe, Adamts3 and Adamts14 (Hogan 2017; Potente et al., 2011). Complex morphogenic and cell and Schulte-Merker, 2017; Wang et al., 2020). differentiation processes orchestrate formation of arteries, veins Recent reports have highlighted the importance of actin dynamics and capillaries, as well as of the lymphatic vascular system. in EC during morphogenesis (Gebala et al., 2016; Lamalice et al., 2007; Phng et al., 2015). However microtubules, although crucially 1Integrative Vascular Biology Laboratory, Max-Delbrück Center for Molecular involved in cell division (Belmont et al., 1990), polarity (Siegrist and Medicine in the Helmholtz Association (MDC), Robert-Rössle-Strasse 10, Berlin Doe, 2007) and vesicle transport mechanisms (Vale, 2003), have 2 13125, Germany. DZHK (German Center for Cardiovascular Research), Partner received little attention in vascular biology research. This is despite site, Potsdamer Str. 58, 10785 Berlin, Germany. 3Department of Immunology, Genetics and Pathology, Uppsala University, 752 37 Uppsala, Sweden. 4Vascular their relevance for tumour angiogenesis and vessel maintenance, as Patterning Laboratory, Center for Cancer Biology, VIB, Leuven B-3000, Belgium. microtubule-targeting drugs used for cancer therapy are anti- 5Vascular Patterning Laboratory, Center for Cancer Biology, Department of Oncology, KU Leuven, Leuven B-3000, Belgium. 6Berlin Institute of Health (BIH), angiogenic and/or stimulate the collapse of tumour vasculature Anna-Louisa-Karsch-Straβe 2, 10178 Berlin, Germany. (Hayot et al., 2002; Kruczynski et al., 2006; Schwartz, 2009; Shi et al., 2016; Tozer et al., 2002). The mechanisms behind the selectivity of *Author for correspondence ([email protected]) this phenomenon are not understood. Microtubules are unbranched M.B.d.O., 0000-0002-6635-3963; B.C., 0000-0003-1606-6373; I.G., 0000-0003- cytoskeleton polymers made of α-andβ-tubulin heterodimers that 3297-4208; H.G., 0000-0002-3030-0384 assemble in a directional manner to shape a polarised hollow tube. α- This is an Open Access article distributed under the terms of the Creative Commons Attribution Tubulin carries a tyrosine residue that can be removed post- License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, translationally by the carboxypeptidase Vasohibin 1 (Vash1) distribution and reproduction in any medium provided that the original work is properly attributed. (Aillaud et al., 2017; Nieuwenhuis et al., 2017), in a reaction Handling editor: Steve Wilson referred to as tubulin detyrosination. Tubulin detyrosination is crucial Received 15 July 2020; Accepted 14 January 2021 for neuronal development (Erck et al., 2005), cell division (Barisic DEVELOPMENT 1 RESEARCH ARTICLE Development (2021) 148, dev194993. doi:10.1242/dev.194993 et al., 2015; Liao et al., 2019) and influences vesicle transport (Dunn Injections of MO against vash1 and control MO were performed in et al., 2008). Interestingly, VASH1 was previously identified as a the Tg[fli1ep:EGFP-DCX] zebrafish line, selectively labelling VEGFA-inducible gene in vitro (Abe and Sato, 2001) and is endothelial microtubules by expression of GFP-tagged expressed in the developing endothelium in vivo (Hosaka et al., 2009; microtubule-binding protein doublecortin (Phng et al., 2015). Shibuya et al., 2006; Watanabe et al., 2004). Viral overexpression as Tubulin detyrosination was assessed by antibody staining against well as VASH1 protein administration experiments in vitro and in vivo the glutamate residue of α-tubulin that becomes available after the show that VASH1 inhibits angiogenesis and reduces vessel diameter tyrosine residue is removed by Vash1 (Fig. 2C). Microtubules (Hosaka et al., 2009; Kern et al., 2009). Conversely, Vash1 knockout detected by this antibody are referred to as microtubules with mice exhibited more vascularised tumours (Hosaka et al., 2009) and detyrosinated tubulin (dTyr). Whereas control embryos exhibited a increased angiogenesis in the ear skin injury model (Kimura et al., high intensity array of dTyr in the neural tube, centrosomes, 2009). motoneurons (Fig. 2D,E) and EC (Fig. 2E′, arrowhead), vash1 KD Although these studies demonstrated that VASH1 is necessary for embryos showed strongly diminished labelling of these structures controlling angiogenesis in a tumour and injury onset in vivo, they (Fig. 2F,G). In particular, endothelial labelling was absent following predated the discovery of VASH1 as a microtubule-modifying vash1 KD (Fig. 2G′). Quantification of the ratio between dTyr tubulin enzyme. Thus, the role of microtubule detyrosination and VASH1 in and DCX (endothelial microtubules) signal intensities (Fig. 2H) particular in the context of in vivo angiogenesis remains unknown. confirmed that detyrosination of endothelial microtubules is Here, we use the zebrafish trunk vasculature to characterise significantly reduced in vash1 KD ISVs (dTyr/DCX=0.23±0.15; microtubule detyrosination and the effects of vash1 knockdown mean±s.d.) in comparison with control ISVs (dTyr/DCX=0.32±0.24). (KD) on secondary sprouting and lymphatic development. Using Thus, Vash1 detyrosinates tubulin in zebrafish tissues including the high-resolution live imaging, combined with immunofluorescence endothelium. and gene expression analysis, we find that endothelial tubulin is predominantly detyrosinated in secondary sprouts. vash1 KD causes Vash1 regulates secondary sprouting morphogenic defects in secondary but not primary sprouts, impeding Live-imaging of sprouting angiogenesis in Tg[kdrl-l:ras-Cherry; proper lymphatic development. Mechanistically, vash1 loss-of-