Vegfr Code Reveal Organotypic Roles for the Endothelial Cell Receptor Kdr in Developmental Lymphangiogenesis

Article

Evolutionary Differences in the Vegf/Vegfr Code Reveal Organotypic Roles for the Endothelial Cell Receptor Kdr in Developmental Lymphangiogenesis Graphical Abstract

Authors Adam J. Vogrin, Neil I. Bower, Menachem J. Gunzburg, ..., Steven A. Stacker, Benjamin M. Hogan, Marc G. Achen

Correspondence [email protected] (B.M.H.), [email protected] (M.G.A.)

In Brief Lymphatic vessels display organotypic function and develop in an organ-specific manner. Vogrin et al. find that the zebrafish Kdr receptor is indispensible for craniofacial, but not trunk, lymphangiogenesis whereas Flt4 is essential for the latter. Thus, vascular endothelial growth factor (VEGF) receptor signaling pathways are differentially employed in different tissues to drive developmental lymphangiogenesis.

Highlights d

Kdr is important for lymphatic vascular development

d

Vegf/Vegfr regulation of lymphatic development is spatially dependent

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The Vegf/Vegfr binding code in zebrafish has shifted compared with mice and humans

d

Lymphatic fate throughout the zebrafish embryo is driven by Vegfr signaling

Vogrin et al., 2019, Cell Reports 28, 2023–2036 August 20, 2019 ª 2019 The Author(s). https://doi.org/10.1016/j.celrep.2019.07.055

Cell Reports

Article Evolutionary Differences in the Vegf/Vegfr Code Reveal Organotypic Roles for the Endothelial Cell Receptor Kdr in Developmental Lymphangiogenesis Adam J. Vogrin,1,5 Neil I. Bower,2,5 Menachem J. Gunzburg,3 Sally Roufail,1 Kazuhide S. Okuda,2 Scott Paterson,2 Stephen J. Headey,3,6 Steven A. Stacker,1,4 Benjamin M. Hogan,2,7,8,* and Marc G. Achen1,4,9,* 1Tumour 2Division

Angiogenesis and Microenvironment Program, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia of Genomics of Development and Disease, Institute for Molecular Bioscience, University of Queensland, St. Lucia, QLD 4072,

Australia 3Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC 3052, Australia 4Department of Surgery, Royal Melbourne Hospital, and Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, VIC 3000, Australia 5These authors contributed equally 6Present address: School of Science, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia 7Present address: Organogenesis and Cancer Program, Peter MacCallum Cancer Centre, Melbourne, VIC 3000, Australia 8Present address: Department of Anatomy and Neuroscience, University of Melbourne, Melbourne, VIC 3000, Australia 9Lead Contact *Correspondence: [email protected] (B.M.H.), [email protected] (M.G.A.) https://doi.org/10.1016/j.celrep.2019.07.055

SUMMARY

Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis. INTRODUCTION The lymphatic vasculature is critical for tissue fluid balance, immune function, and the absorption of dietary fat, and plays important roles in human diseases such as cancer metastasis and lymphedema (Alitalo, 2011; Stacker et al., 2014). This vasculature consists of distinct types of lymphatic vessels such as initial lym-

phatics, which absorb tissue fluid, and precollecting lymphatics, lymphatic collectors, and lymphatic ducts responsible for transport of lymph. Much progress has been made over the past decade in defining molecular signaling pathways controlling lymphatic development in the embryo (Koltowska et al., 2013). However, the ways in which these molecular mechanisms vary during organotypic lymphatic development, which generates lymphatic networks with distinct structure and function in different tissues and organs such as skin, lymph nodes, and small intestine (Petrova and Koh, 2018), require further study. Vascular endothelial growth factors (VEGFs) are critical for development of the blood and lymphatic vasculatures and are important in human disease (Alitalo, 2011; Shojaei and Ferrara, 2007; Stacker et al., 2014). For example, the angiogenic protein VEGFA is a key therapeutic target in ocular indications and cancer. VEGFC and VEGFD promote growth of new lymphatic vessels (lymphangiogenesis), lymphatic vessel remodeling, and angiogenesis, and are candidate therapeutic targets in cancer and cardiovascular medicine (Stacker et al., 2014; Zheng et al., 2014). VEGFA is thought to promote angiogenesis by activating the endothelial cell surface receptor VEGFR2 (Millauer et al., 1993). Human VEGFC and VEGFD activate both VEGFR2 and VEGFR3 (Achen et al., 1998; Joukov et al., 1996), and promote lymphangiogenesis and angiogenesis in mammalian models (Baldwin et al., 2005; Karkkainen et al., 2004; Rissanen et al., 2003). VEGFC and VEGFD require proteolytic processing to activate their receptors, involving removal of N- and C-terminal propeptides from a central receptor-binding domain (Joukov et al., 1997; Stacker et al., 1999). Human VEGFR3 can also be proteolytically processed, involving cleavage in the extracellular region and formation of inter-subunit disulfide bonds between the two resulting polypeptides (Leppa¨nen et al., 2013; Pajusola et al., 1994), but the functional significance of this processing is not known. VEGFR3 signaling is considered apical in developmental lymphangiogenesis. In early development, complete loss of VEGFR3

Cell Reports 28, 2023–2036, August 20, 2019 ª 2019 The Author(s). 2023 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

in mice results in cardiovascular failure at embryonic day (E) 9.5, prior to the emergence of the lymphatic vasculature (Dumont et al., 1998). Heterozygous mutations in the gene for VEGFR3 result in lymphatic vascular defects in mice and humans (Irrthum et al., 2000; Karkkainen et al., 2001), and loss of the VEGFR3 ligand VEGFC leads to lymphatic defects and decreased lymphatic sprouts in mice (Karkkainen et al., 2004). VEGFR3 has been shown to be important in the tip cells of lymphatics and blood vessels, thus playing a key role in vascular patterning and sprouting in mice (Dumont et al., 1998; Tammela et al., 2008). VEGFR2 signaling has also been implicated in the biology of lymphatic vessels as it contributes to VEGFC-induced downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) signaling in lymphatic endothelial cells (LECs) in vitro (Deng et al., 2015). However, loss of VEGFR2 in mice leads to lethality due to defects in the development of hematopoietic and endothelial cells at E8.5, prior to lymphatic specification, thus precluding analysis of lymphangiogenesis (Shalaby et al., 1995). Some reports have shown an influence of VEGFR2 during lymphangiogenesis in vivo in mice (Saharinen et al., 2010; Wirzenius et al., 2007), and deletion of VEGFR2 in vasculature reduced lymphatic vessel density in the developing dermis (Dellinger et al., 2013). However, it has remained unclear to what degree VEGFR2 contributes to lymphangiogenesis in the developing embryo and collaborates or acts redundantly with VEGFR3. In fish, birds, and marsupials, there are four Vegf receptors, with the presence of the ohnologues kdr and kdrl complicating and potentially diversifying the Vegf/Vegfr code by comparison with placental vertebrates (Bussmann et al., 2007). The encoded zebrafish Kdr and Kdrl receptors are both expressed throughout the vasculature and both bind Vegfa (Bahary et al., 2007). In zebrafish, primary angiogenesis occurs from approximately 22 h post fertilization (hpf) when arterial sprouts develop from the dorsal aorta to give rise to the intersegmental vessels in a process that is dependent on Vegfa-driven signaling (Bahary et al., 2007; Covassin et al., 2006; Lawson and Weinstein, 2002b). Zebrafish kdrl mutants form reduced numbers of intersegmental vessels, while loss of kdr has no impact on the formation of arterial intersegmental vessels. However, loss of both kdrl and kdr leads to a complete loss of these vessels (Covassin et al., 2006). Secondary angiogenesis, which proceeds from approximately 32 hpf, involves angiogenic sprouting of the cardinal vein and gives rise to intersegmental veins and parachordal LECs at the horizontal myoseptum (Hogan and Schulte-Merker, 2017; Isogai et al., 2003). These LECs subsequently proliferate and migrate to form the trunk lymphatic network (Cha et al., €chler et al., 2006; Yaniv et al., 2006). In the craniofacial 2012; Ku region of the embryo, LECs emerge from the common cardinal vein and fuse with LECs that emerge from the primary head sinus to form a complex facial lymphatic network; meanwhile, a population of mural LECs also forms around the brain (Astin et al., 2014; Bower et al., 2017a; Okuda et al., 2012; van Lessen et al., 2017; Venero Galanternik et al., 2017). Both vegfc and vegfd zebrafish mutants show reductions in, and double vegfc, vegfd mutants show major or complete loss of, craniofacial lymphatic development (Bower et al., 2017a, 2017b). Zebrafish mutants lacking Flt4 (the homolog of human VEGFR3) show a complete block in trunk lymphangiogenesis and specification

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of trunk LECs (with no induction of the LEC transcription factor Prox1), but they do not display a complete loss of craniofacial lymphangiogenesis (Shin et al., 2016). We have previously shown that zebrafish Vegfd can bind Kdr but not Kdrl (Bower et al., 2017b); however, it was assumed that the contribution to facial lymphangiogenesis by zebrafish Vegfd occurs via activation of the lymphangiogenic zebrafish receptor Flt4. Here, we report the proteolytic processing, receptor-binding capabilities, and genetic interactions of key zebrafish Vegf ligands and their receptors. Coupled with mutant studies of kdr, our observations uncover an unexpected role for Kdr signaling in driving craniofacial lymphangiogenesis. We show that evolutionary differences occur at the level of the Flt4, Kdr, and Kdrl receptors rather than their ligands. Our findings identify a definitive, spatially dependent, role for a VEGFR2 homolog in driving developmental lymphangiogenesis and LEC specification, and provide a biochemical roadmap to better appreciate the role of evolutionary change in the Vegf/Vegfr code for vascular development. Importantly, we also define distinct molecular mechanisms promoting lymphangiogenesis in the craniofacial region and trunk of zebrafish, thus providing key insight into the control of organotypic modes of lymphangiogenesis. RESULTS Zebrafish Vegfc and Flt4 Are Proteolytically Processed Similarly to Their Human Homologs We previously showed that the proteolytic processing of zebrafish Vegfd is similar to its human homolog (Bower et al., 2017b). However, our current understanding of the processing of zebrafish Vegfc and Flt4 is limited, so we tested if they are proteolytically processed to better understand the biosynthesis of these proteins and to guide our choice of protein constructs for receptor-binding studies. Alignment of human and zebrafish VEGFC identified five residues on either side of two sites at which the N-terminal propeptide can be cleaved in human VEGFC (these sites are somewhat conserved in zebrafish) and five residues preceding the C-terminal propeptide cleavage site that are completely conserved (Figure 1A). Immunoprecipitation of a full-length zebrafish Vegfc tagged with a N-terminal FLAG octapeptide (designated zVegfcFULL-N-FLAG) followed by anti-FLAG western blotting revealed the presence of similar proteolytic processing to that which occurs for human VEGFC (Figure 1B) with bands at
53 kDa (full-length Vegfc) and
35 kDa (N-terminal propeptide and central domain), as well as a weaker band at
10 kDa (N-terminal propeptide). These findings indicate that zebrafish Vegfc can be proteolytically processed in a similar fashion to human VEGFC, with removal of propeptides, which could lead to generation of a mature form. Human VEGFR3 is cleaved between residues 472 and 473 in the fifth immunoglobulin (Ig)-like domain of the extracellular region, as identified from the crystal structure of the protein (Leppa¨nen et al., 2013). Alignment with the zebrafish Flt4 sequence indicates some, but not a high degree of conservation (Figure 1C). We expressed the entire Flt4 extracellular domain fused at the C terminus to the Fc region of mouse IgG (zFlt4-Fc) and performed immunoprecipitation followed by western blotting targeting the mouse Fc region (Figure 1C). Two bands were detected

A

Human VEGF-C Zebrafish Vegfc

98 NLNSRTEETIKFAAAHYNT 116 87 STETRSEEA-SFAAAFINL 104

223 SIIRRSLPAT 232 210 SIIRRALTEC 219

Figure 1. Vegfc and Flt4 from Zebrafish Can Be Proteolytically Processed

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Co nt ro l zV eg fc Fu ll Co nt ro l zV eg fc Fu ll

(A) Alignment of amino acid sequences of human VEGFC and zebrafish Vegfc focusing on where Nand C-terminal propeptides (N-pro and C-pro, respectively) can be cleaved from the central N-pro VHD C-pro VEGF homology domain (VHD) in the human protein. Arrows indicate proteolytic cleavage sites. C B (B) Analysis of zebrafish Vegfc produced by Human VEGFR-3 465 PCKM-FAQRSLRRRQQQD 481 HEK293T cells. A full-length form of Vegfc tagged Zebrafish Flt4 492 PCDLERTRRALRRRGGRD 509 with the FLAG octapeptide at the N terminus, zVegfcFULL-N-FLAG (zVegfcFull), was transiently expressed in HEK293T cells, precipitated from Cyto TM 62 conditioned media with anti-FLAG antibody and 1 2 3 4 5 6 7 F N-pro VHD C-pro analyzed by anti-FLAG western blotting (reducing 49 conditions). Control indicates results with condi38 tioned media of cells transfected with expression F N-pro VHD vector lacking DNA encoding Vegfc. Two different 28 exposures of the same blot are shown. Positions of molecular weight markers are shown to the left Unprocessed zFlt4-Fc 198 14 (kDa) and schematic representations of detected Fc F N-pro 1 2 3 4 5 6 7 98 proteins are shown to the right. F denotes FLAG 6Fc tag and dotted lines indicate where images were 5 6 7 Short Long 62 Cleaved zFlt4-Fc spliced to remove irrelevant tracks. Exposure Exposure (C) Top: alignment of sequences of human IP: Anti-FLAG VEGFR3 and zebrafish Flt4 where proteolytic IP: Protein A-Sepharose Blot: Anti-FLAG Blot: Anti-mouse IgG processing occurs in the human protein. Arrow indicates proteolytic cleavage site. The schematic of VEGFR3 shows the seven Ig-like domains of the extracellular region (blue line) and the transmembrane (TM) and cytoplasmic (Cyto) domains. Bottom: a protein consisting of the entire zebrafish Flt4 extracellular domain and, at the C terminus, the Fc region of mouse IgG (zFlt4-Fc) was transiently expressed in HEK293T cells, precipitated from conditioned media with protein A-Sepharose and analyzed by western blotting with antibodies to mouse IgG (reducing conditions). Control indicates results with conditioned media of cells transfected with expression vector lacking DNA encoding zFlt4-Fc. The
150-kDa band is the expected size of zFlt4-Fc. The
75-kDa band is the expected size of a form of zFlt4-Fc, containing part of Ig-like domain 5, Ig-like domains 6 and 7, and Fc, that arises due to proteolytic cleavage in the fifth Ig-like domain. Molecular weight markers (kDa) are shown to the left. All precipitations and western blots were conducted three times using fresh media, with results similar to those shown in (B) and (C).

corresponding to full-length Flt4-Fc (
150 kDa) and a shorter form in which cleavage had occurred in Ig-like domain five of the Flt4 extracellular region (
75 kDa). Thus, zebrafish Flt4 can be proteolytically processed similarly to human VEGFR3. Identification and Validation of Key Vegf/Vegfr Binding Specificities and Functions in Zebrafish Our understanding of the specificity of the interactions between Vegf ligands and receptors in zebrafish is incomplete, so we monitored this in receptor-binding studies. Given that zebrafish Vegfc can be proteolytically processed, and that the central VEGF homology domain (VHD) in the human protein is responsible for binding VEGF receptors, we used a mature form of the zebrafish protein, consisting of the central VHD with an N-terminal FLAG tag (zVegfcDNDC-FLAG), for receptor-binding studies. Our finding that zebrafish Flt4 can also be proteolytically processed led us to employ two Flt4 constructs—zFlt4-Fc (described above) and a Fc-fusion construct with only the outer three Ig-like domains of Flt4 (zFlt4(1-3)-Fc), which was used because we were unsure how proteolytic processing of Flt4 might interfere with ligand binding, and the outer three Ig-like domains of human VEGFR3 are sufficient for binding VEGFC and VEGFD (Leppa¨nen et al., 2011, 2013). In addition, we used the following recombinant forms of zebrafish Vegfs and their receptors: an N-terminally FLAG-tagged 121-amino acid isoform of zebrafish Vegfaa (zVegfaa121-FLAG) homologous to human

VEGFA121 (Bahary et al., 2007), a mature form of zebrafish Vegfd with an N-terminal FLAG tag (zVegfdDNDC-FLAG) (Bower et al., 2017b), Fc fusion constructs containing the entire extracellular domains of Kdr (zKdr-Fc) or Kdrl (zKdrl-Fc) (Bower et al., 2017b), or the outer three Ig-like domains of Flt1 (zFlt1(1-3)-Fc), which was used because we found that a Fc construct with the entire Flt1 extracellular domain was expressed poorly in various cell lines, and the outer three Ig-like domains of human VEGFR1 are sufficient for ligand binding (Markovic-Mueller et al., 2017). Vegf ligands were ‘‘pulled down’’ using Fc fusion constructs and detected by anti-FLAG western blotting. This revealed that zVegfaa121-FLAG bound zKdr-Fc, zKdrl-Fc, and zFlt1(1-3)-Fc but not zFlt4(1-3)-Fc or zFlt4-Fc (Figure 2A). We tested for further evidence of these interactions using genetic epistasis analysis by mRNA injections into zebrafish embryos that had transgene-labeled blood vasculature. We first analyzed injected embryos at a stage prior to lymphangiogenesis, and found that injection of mRNA encoding either of the Flt4-Fc constructs did not reduce formation of primary (blood vessel) sprouts (a process driven by Vegfa [Bahary et al., 2007]) while expression of a soluble form of Flt1 led to a reduction in primary sprout formation and expression of zKdrl-Fc caused complete loss of primary sprouts (Figures S1A and S1C). These findings are broadly consistent with the pulldown studies. Our pulldown studies also showed that zVegfcDNDC-FLAG bound both

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zV eg zV faa eg 12 1 f zV c'N eg 'C f Co d'N nt ro 'C l zV eg zV faa eg 12 zV fc'N 1 eg ' Co fd' C nt N ' ro C l zV eg f zV aa1 21 eg zV fc'N eg ' Co fd' C nt N ' ro l C

zV eg zV faa1 eg 21 zV fc'N eg ' Co fd' C nt N ' ro l C zV eg f zV aa1 2 eg fc 1 ' zV eg N'C Co fd' nt N ' ro C l zV eg f zV aa12 eg 1 zV fc' eg N' Co fd' C nt N ' ro l C

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Figure 2. Interactions of Zebrafish Vegf Ligands with Receptors (A) Analysis of ligand binding by receptor pull-down. zVegfaa121-FLAG (zVegfaa121), zVegfcDNDC-FLAG (zVegfcDNDC), and zVegfdDNDC-FLAG (zVegfdDNDC) were precipitated from conditioned media of transiently transfected HEK293T cells using zFlt1(1-3)-Fc, zKdr-Fc, zKdrl-Fc, zFlt4-Fc, zFlt4(1-3)-Fc, and the Fc domain of mouse IgG. Conditioned media of HEK293T cells transiently transfected with expression vector lacking DNA encoding Vegf ligands was negative control (Control). Ligands were detected by anti-FLAG western blotting (reducing conditions). Input levels of ligands and receptors in all precipitations were controlled as described in the STAR Methods, and all blots were conducted in parallel. Molecular weight markers (in kDa) are indicated on the left, and dotted lines indicate where images were spliced to remove irrelevant tracks. Expected sizes for subunits of these Vegf family ligands are 20–26 kDa. All precipitations and western blots were conducted at least three times using fresh media, with results similar to those shown here. (B) Confocal micrographs of 30-hpf Tg(fli1a:EGFP) zebrafish embryos showing formation of arterial intersegmental vessels (aISVs) in uninjected control and turning of aISVs (arrowheads) following injection of vegfc or vegfd mRNA. The turning of aISVs following injection of vegfc, but not vegfd, mRNA is rescued by coinjection of mRNA for zFlt4(1-3)-Fc. In contrast, co-injection of mRNA for zFlt1-Fc does not rescue the turning of aISVs induced by either vegfc or vegfd mRNA. Quantitation of turned aISVs in response to injection of different mRNAs is shown in the graph (n = 33 embryos for all study groups; ***p < 0.0001). (C) Confocal micrographs of 54-hpf Tg(fli1a:EGFP) zebrafish embryos showing the development of parachordal LECs (PLs) in uninjected controls and embryos injected with mRNA for zFlt4(1-3)-Fc or zKdrl-Fc. The regions in dotted rectangles are shown at higher magnification in (C0 ) with arrowheads highlighting PLs in uninjected embryos and those injected with mRNA for zKdrl-Fc, and asterisks indicating where PLs are absent in embryos injected with mRNA for zFlt4(1-3)-Fc. Scale bars in (B) and (C) represent 100 mm and apply to all images. (D) Quantification of PLs reveals a significant reduction following injection of mRNA for zFlt4(1-3)-Fc (n = 37 embryos) or zKdrl-Fc (n = 28) compared to uninjected controls (n = 32). ****p < 0.0001. Data are represented as the mean ± SEM.

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zFlt4-Fc and zFlt4(1-3)-Fc, as well as zKdr-Fc, but bound neither zKdrl-Fc nor zFlt1(1-3)-Fc (Figure 2A). This was supported by the finding that bilateral turning of arterial intersegmental blood vessels (aISVs) induced by injection of vegfc mRNA in zebrafish embryos was blocked by co-injection of mRNA for zFlt4(1-3)-Fc but not soluble Flt1 (Figure 2B). Furthermore, when we examined later secondary sprouting (a process that will give rise to the earliest lymphatics and is dependent on Vegfc), we found sprouting was blocked by injection of mRNA for zFlt4(1-3)-Fc but not zKdrl-Fc (Figures 2C, 2D, S1B, S1D, and S1E). zVegfdDNDC-FLAG bound zKdr-Fc but not zKdrlFc in pull-down studies, as reported previously (Bower et al., 2017b), but did not bind zFlt1(1-3)-Fc nor, surprisingly, did it bind either of the Flt4 constructs (Figure 2A). The failure of Vegfd to bind the Flt4-Fc constructs contrasts with previously reported high-affinity binding of mature human VEGFD by human VEGFR3 (Achen et al., 1998). Our pull-down studies, involving versions of zVegfc and zVegfd consisting essentially of the VHD, demonstrated that binding sites for zKdr in both ligands, and for zFlt4 in zVegfc, are located in the central VHD of these ligands. Surface plasmon resonance was used as an alternative approach to monitor ligand-receptor interactions and revealed receptor-binding specificities entirely consistent with the pull-down experiments; zVegfcDNDC-FLAG bound zFlt4-Fc whereas zVegfdDNDC-FLAG and zVegfaa121-FLAG did not (Figure 3A). Single-cycle kinetics were used to derive the binding constants of the interaction of zVegfcDNDC-FLAG with zFlt4-Fc (Figure 3A). The equilibrium dissociation constant was 2.7 nM. Thus, the high-affinity interaction of the zebrafish Vegfc and Flt4 constructs is very similar to the 5.6 nM value for human mature VEGFC interacting with the extracellular domain of VEGFR3 previously derived by surface plasmon resonance (Leppa¨nen et al., 2013). To rule out the possibility that the N-terminal FLAG tag in zVegfdDNDC-FLAG interfered with binding to the Flt4-Fc constructs, we generated two alternative forms of mature zebrafish Vegfd: (1) zVegfdDNDC-FLAG-Cterm, which was similar to zVegfdDNDC-FLAG except that the FLAG tag was incorporated at the C terminus, not the N terminus, and (2) zVegfdDNDCLong-FLAG, which was similar to zVegfdDNDC-FLAG except that an extra 12 amino acids from Vegfd were included C terminal to the FLAG tag (Figure 3B). Both zVegfdDNDC-FLAG-Cterm and zVegfdDNDC-Long-FLAG showed the same receptor specificity as zVegfdDNDC-FLAG in pull-down experiments, binding zKdrFc but not zFlt4-Fc or zKdrl-Fc (Figure 3C). The receptor interactions of zebrafish Vegfd were further explored in vivo by injection of vegfd mRNA in zebrafish embryos, which led to a primary angiogenic sprouting phenotype that was not blocked by co-expression of zFlt4-(1-3)-Fc (Figure 2B), likely due to the inability of Vegfd to bind Flt4. Further, injection of vegfd mRNA partially rescued the block in primary angiogenesis caused by expression of zKdrl-Fc (Figures 3D and 3E), which is consistent with the findings of Rossi et al. (2016), demonstrating that Vegfd is capable of rescuing the loss of arterial intersegmental vessels in vegfaa zebrafish mutants, an observation consistent with the capacity of Vegfd to bind Kdr (Bower et al., 2017b).

Overall, our receptor binding studies demonstrated that zebrafish Vegfc binds both Kdr and Flt4 but not Flt1 or Kdrl. This is broadly consistent with the receptor binding specificity of human VEGFC, which binds VEGFR2 and VEGFR3 but not VEGFR1 (Joukov et al., 1997). We also show that zebrafish Vegfd binds Kdr but not Flt4, Kdrl, or Flt1. The inability of Vegfd to bind Flt4 contrasts with human VEGFD, which binds both VEGFR2 and VEGFR3, but not VEGFR1 (Achen et al., 1998). Zebrafish Vegfaa bound to Flt1, Kdr, and Kdrl, but not Flt4, which is similar to human VEGFA, which binds VEGFR1 and VEGFR2 but not VEGFR3 (Kowanetz and Ferrara, 2006). Evolutionary Divergence in VEGFR3 Binding by VEGFD Is Primarily Determined by the Receptor The distinct ligand-binding capabilities of Kdr and Kdrl, combined with the inability of Flt4 to bind Vegfd in zebrafish, is likely due to the evolutionary differences among vertebrates causing diversification in the VEGF-VEGFR code during development. The biochemical nature of this change in the code could be due to species-specific changes in the ligand, the receptor, or both. To further understand this, we tested the capacity of zebrafish Vegfd to bind human VEGFR3 and of human VEGFD to bind zebrafish Flt4. For these studies we employed a N-terminally FLAG-tagged version of mature human VEGFD (VEGF-DDNDC) that was previously shown to bind human VEGFR2 and VEGFR3 (Achen et al., 1998), and receptor-Ig fusion proteins containing the entire extracellular domains of human VEGFR2 or VEGFR3 with the Fc region of human IgG at the C-terminal end of the proteins. Pull-down experiments showed that human VEGFD, like zebrafish Vegfd, did not bind zebrafish Flt4 (Figure 3F), whereas both of these ligands bound human VEGFR3 (Figure 3G). These two ligands also bound zebrafish Kdr and human VEGFR2 but not Kdrl or Flt1 (Figures 3F and 3G). We also showed that zebrafish Vegfc, like human VEGFC, binds both human VEGFR2 and VEGFR3 (Figure 3G). Taken together, these findings indicate that the inability of both human and zebrafish VEGFD to bind zebrafish Flt4, whereas both ligands bind human VEGFR3, likely reflects differences between the structures of these two receptors. Kdr Regulates Craniofacial Lymphangiogenesis in Zebrafish Zebrafish vegfd mutants display defects in craniofacial lymphangiogenesis and flt4 mutants are capable of some craniofacial lymphangiogenesis despite loss of all trunk lymphangiogenesis (Bower et al., 2017b; Shin et al., 2016). As our biochemical findings indicate that Vegfd can only bind Kdr, this suggests a role for Vegfd/Kdr signaling in lymphangiogenesis. Overall, we postulated that the presence of the two functional ohnologues (Kdr/Kdrl) in teleost fish might provide us with a capacity to investigate the role of VEGFR2 signaling in lymphangiogenesis. Therefore, we generated a CRISPR mutant zebrafish line harboring a 2-bp insertion at amino acid 175 in exon 5 of the kdr locus, introducing a premature stop codon at amino acid 181, which is located in the second Ig-like domain of the extracellular region. Thus, any truncated Kdr protein produced would consist of only the first, and a truncated second, Ig-like domain and would lack ligand-binding capacity as both Ig-like domains

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two and three are required for ligand binding by human VEGFR2 (Fuh et al., 1998; Leppa¨nen et al., 2010). To validate the loss-of-function mutation and to test the function of zVegfd in vivo, we injected vegfd mRNA into embryos from an in-cross of heterozygous (kdruq38bh) fish and quantified the number of turned aISVs. Subsequent genotyping revealed that all embryos displaying zVegfd-induced turned aISVs were wild-type or heterozygous for kdruq38bh, whereas homozygous mutant embryos did not exhibit turning (Figures 4A and 4B), demonstrating that zVegfd drives this phenotype through Kdr in vivo. Next, heterozygous carriers (kdruq38bh) were in-crossed and the number of LECs in the thoracic duct quantified; however, we found no difference between wild-type sibling and mutant embryos (Figures 4C, 4C0 , and 4G). Additionally, examination of the intersegmental blood vessels revealed no difference in the formation of intersegmental arteries (ISAs) or intersegmental veins (ISVs) between mutant and wild-type siblings (Figures 4C, 4C0 , and 4H). However, quantification of transgene-labeled nuclei present in facial lymphatic vessels revealed significantly fewer LECs in the medial (MFL), lateral (LFL), and otolithic (OLV) facial lymphatics in kdr mutants when compared with wild-type and heterozygous siblings (Figures 4D–4F and 4I– 4K). We also observed a marked reduction in early mural LECs (also known as fluorescent granular perithelial cells [FGPs] or brain LECs) (Bower et al., 2017a; van Lessen et al., 2017; Venero Galanternik et al., 2017) of the larval brain (Figures 4D–4F). Consistent with roles for vegfd and kdr in rostral-craniofacial lymphangiogenesis, in situ hybridization revealed expression of kdr as well as flt4 at 36 hpf in the regions where lymphatic precursors emerged from the common cardinal vein and the primary head sinus (Figures 4L, 4M, and S2A–S2F). Additionally, analysis of a vegfd BAC transgenic line that reports vegfd transcription (TgBAC(vegfd:eYFP)uq42bh) revealed expression of vegfd at 36

hpf in close proximity to the region where we observed kdr and flt4 expression, similar to the in situ hybridization expression pattern described previously (Astin et al., 2014) (Figures 4N and S2G–S2I). At 60 and 72 hpf, vegfd expression was observed in close proximity to the emerging otolithic lymphatic vessel, lymphatic branchial arches, and the region where mural LECs emerged from the choroidal vascular plexus (Figures 4O and S2J–S2M) (Bower et al., 2017a; Okuda et al., 2012). Although our biochemical analysis demonstrated that Kdr binds Vegfa, similarly to our observations in the trunk, we did not identify any defects in the formation of the cranial blood vasculature by micro-angiography (Figures S3A and S3B). Together these findings identified that Kdr is essential for rostral, craniofacial lymphangiogenesis in zebrafish development but is dispensible for caudal, trunk lymphangiogenesis, and whole organism blood vascular angiogenesis, emphasizing the spatially dependent nature of its role in regulating lymphatic development. Given that Vegfc and Vegfd compensate for each other in both facial and trunk lymphangiogenesis (Astin et al., 2014; Bower et al., 2017b) and that Vegfd binds only to Kdr, we next hypothesized that Kdr and Flt4 may compensate for each other in lymphangiogenesis more broadly. We generated double mutants for kdruq38bh and the hypomorphic flt4hu4602 alleles (Hogan et al., 2009b) and assessed lymphangiogenesis in the developing trunk with single-cell resolution. We found that in flt4hu4602 heterozygous embryos, which develop a thoracic duct with reduced LEC numbers, loss of kdr significantly enhanced the phenotype leading to a complete loss of the thoracic duct (Figures 4P–4V). These data demonstrate that while Kdr is dispensible in the trunk, it can compensate for haploinsufficiency of Flt4 in trunk lymphangiogenesis. Overall, our findings show that Kdr is differentially involved in controlling lymphangiogenesis in the craniofacial region compared to the trunk.

Figure 3. Interactions of Zebrafish Vegfd with Receptors, and Cross-Species Ligand-Receptor Interactions (A) Analysis of ligand interactions with zFlt4-Fc by surface plasmon resonance. zVegfdDNDC-FLAG (green line), zVegfaa121-FLAG (blue), and zVegfcDNDCFLAG (red) were injected in a 3-fold increasing concentration series from 4.1 to 1000 nM across captured control Fc (top graph) and captured zFlt4-Fc (bottom graph). Black line denotes fit to a 1:1 model for binding of zVegfcDNDC-FLAG to zFlt4-Fc. Kinetic parameters of zVegfcDNDC-FLAG binding to zFlt4-Fc are shown in the table. Values are means and standard deviations of fits to the 1:1 binding model, for three independent experiments. (B) Schematic maps of alternative forms of mature zebrafish Vegfd: zVegfdDNDC-FLAG (zVegfdDNDC), VegfdDNDC-FLAG-Cterm (VegfdDNDC-Cterm), and zVegfdDNDC-Long-FLAG (zVegfdDNDC-Long). zVegfdDNDC-Long-FLAG has 12 extra residues at the N terminus of the VHD compared to other constructs. F denotes FLAG. (C) Analysis of receptor binding by alternative forms of mature zebrafish Vegfd. Ligands were precipitated from conditioned media of transfected HEK293T cells using receptor-Fc constructs indicated under each blot. Conditioned media of cells transfected with expression vector lacking DNA for ligands was negative control (Control). Ligands were detected by anti-FLAG western blotting (reducing conditions). Input levels of ligands and receptors in all precipitations were controlled as described in the STAR Methods, and all blots were conducted in parallel. Molecular weight markers (kDa) are indicated and expected sizes for ligand subunits are 20–26 kDa. Dotted line indicates where image was spliced to remove irrelevant track. (D and E) At 30 hpf, embryos injected with mRNA for zKdrl-Fc have fewer arterial intersegmental vessels (aISVs) than uninjected embryos, which can be partially rescued by co-injection of vegfd mRNA (D), quantified in (E) (n = 32 embryos for uninjected, n = 25 for zKdrl-Fc, and n = 34 for zKdrl-Fc + Vegfd; ****p < 0.0001). Scale bar in (D) represents 100 mm and applies to all images. (F) Binding of human VEGFD to zebrafish receptors. Human VEGFDDNDC (hVEGFDDNDC) was precipitated with constructs indicated under blots and detected by anti-FLAG western blotting. Input levels of ligands and receptors in all precipitations were controlled as described in the STAR Methods, and all blots were conducted in parallel. Media from cells transfected with expression vector lacking ligand DNA was negative control (Control), and zVegfaa121-FLAG (zVegfaa121) and zVegfcDNDC-FLAG (zVegfcDNDC) were positive controls. Expected sizes for ligand subunits are 20–26 kDa. (G) Binding of zebrafish Vegfd to human receptors. zVegfdDNDC-FLAG (zVegfdDNDC) was precipitated using human VEGFR2-Fc (hVEGFR2-Fc), human VEGFR3-Fc (hVEGFR3-Fc), or Fc of mouse IgG, and detected by western blotting. Input levels of ligands and receptors in all precipitations were controlled as described in the STAR Methods, and all blots were conducted in parallel. Negative control (Control) was as for (F), and hVEGFDDNDC and zVegfcDNDC were positive controls. Molecular weight markers (kDa) are indicated. All precipitations and western blots in (C), (F), and (G) were conducted at least three times using fresh media, with results similar to those shown here. Data are represented as the mean ± SEM.

Cell Reports 28, 2023–2036, August 20, 2019 2029

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Figure 4. Kdr Interacts with zVegfd In Vivo and Regulates Craniofacial Lymphangiogenesis but Not Development of the Thoracic Duct (A) Confocal images of 30-hpf Tg(fli1a:nEGFP) embryos injected with vegfd mRNA showing turned aISVs (arrowheads) in wild-type but not in kdr homozygous mutant siblings. (B) Quantification of turned aISVs in wild-type (n = 39), kdruq38bh heterozygous (n = 42), and homozygous mutant (n = 15) embryos at 30 hpf.

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2030 Cell Reports 28, 2023–2036, August 20, 2019

Early Embryonic Lymphatic Endothelial Cell Specification Is Induced by Vegfr Signaling We, and others, have recently shown that LEC specification, as indicated by expression of the key transcription factor Prox1, is driven by Vegfc/Flt4 signaling in an Erk-dependent manner in the zebrafish trunk (Baek et al., 2019; Koltowska et al., 2015; Shin et al., 2016). Loss of Flt4 leads to a loss of the earliest detectable Prox1 expression in the trunk, as assessed by immunofluorescence studies; however, in flt4 null mutants Prox1 expression in the facial lymphatic progenitors was still observed (Shin et al., 2016). This suggests that different mechanisms induce differentiation of the lymphatic vascular lineage in different vessel beds. To determine if Kdr and Flt4 may be required together for facial lymphangiogenesis, we first quantified the number of nuclei contributing to facial lymphatic vessels in kdruq38bh, flt4hu4602 double-mutant embryos. Strikingly, some facial LECs, including in the prominent rostral lymphatic structure (RLS) that forms in flt4 null mutants (Shin et al., 2016), completely failed to form and sprout in kdruq38bh, flt4hu4602 double-mutant embryos (Figures 5A–5H). Interestingly, our analyses of kdruq38bh, flt4hu4602 double heterozygotes did not reveal any genetic interaction between kdr and flt4 in these endothelial beds (Figures 5B and 5E–5G). These findings further emphasize that Kdr and Flt4 are differentially involved in controlling lymphangiogenesis in the craniofacial region compared to the trunk. To observe the very earliest step in lymphangiogenesis, which involves the specification of LECs as labeled by Prox1 expression, we examined Prox1 in the common cardinal vein and primary head sinus from which the facial lymphatics derive (Figure S3C). Consistent with previous reports (Shin et al., 2016), flt4hu4602 mutants had mildly reduced numbers of Prox1-positive cells in both of these anatomical structures. In kdruq38bh mutants, Prox1-positive endothelial cells were slightly reduced in the common cardinal vein and significantly reduced in the primary head sinus. However, kdruq38bh, flt4hu4602 double-mutant em-

bryos had a vast reduction in Prox1-positive endothelial cells in the common cardinal vein and, in some cases, a complete loss in the primary head sinus (Figures 5I–5N). These findings emphasize the central importance of both Flt4 and Kdr for LEC specification in the zebrafish embryo. DISCUSSION This study assessed signaling mechanisms of zebrafish Vegf ligands by monitoring the capacity of these proteins to interact with zebrafish Vegf receptors known to play critical roles in angiogenesis and lymphangiogenesis (these interactions and roles are summarized in Figures 5O and 5P). We demonstrated that zebrafish Vegfc can be proteolytically processed to remove the N- and/or C-terminal propeptides, as shown previously for zebrafish Vegfd (Bower et al., 2017b) and human VEGFC and VEGFD (Joukov et al., 1997; Stacker et al., 1999). This processing is required for high-affinity binding of VEGF receptors in mammals. Hence, the proteases that process and activate Vegfc and Vegfd must be important regulators of vascular development in zebrafish. Proteases responsible for this processing in mammals include ADAMTS3 (A disintegrin and metalloproteinase with thrombospondin motifs-3) and proprotein convertases (Jeltsch et al., 2014; Siegfried et al., 2003). Further, the CCBE1 protein promotes co-localization of human pro-VEGF-C with ADAMTS3 (Jha et al., 2017), and zebrafish Ccbe1 has important roles in lymphangiogenesis and vascular sprouting in embryos (Hogan et al., 2009a; Le Guen et al., 2014). We showed that zebrafish Flt4 can also be proteolytically processed in a similar fashion to its human homolog (Leppa¨nen et al., 2013; Pajusola et al., 1994) but the functional significance of this processing, and the enzyme(s) involved, are unknown. Our findings that mature zebrafish Vegfc bound Kdr and Flt4, but not Kdrl or Flt1, are broadly consistent with human VEGFC. which binds VEGFR2 and VEGFR3, but not VEGFR1 (Joukov

(C and C0 ) Analysis of thoracic duct (TD) development. Confocal images of 5-dpf Tg(fli1a:nEGFP);Tg(5.2lyve1b:dsRed) transgenic embryos produced by incrossing kdruq38bh carriers. TD development in kdruq38bh mutant embryos (C0 ) is equivalent to wild-type (C) siblings. Red denotes Lyve1-positive lymphatics and green denotes Fli1-positive nuclei. (D–F) Analysis of facial lymphatic development. Images showing the facial region of 5-dpf Tg(fli1a:nEGFP);Tg(5.2lyve1b:dsRed) transgenic embryos. Genotypes in (D) and (D0 ) are kdr+/+, (E) and (E0 ) kdr+/, and (F) and (F0 ) kdr/. Red denotes Lyve1-positive lymphatics and green denotes Fli1-positive nuclei. White arrow indicates mural LEC loop and white asterisk denotes its absence. (D0 –F0 ) lyve1b:dsRed only; white anatomical structures are Lyve1-positive lymphatics. Yellow arrows indicate normal lateral facial lymphatics (LFLs), medial facial lymphatics (MFLs), and otolithic facial lymphatics (OLVs). Yellow asterisks indicate where there are reductions in LEC numbers in these vessels. (G) There was no significant difference in the number of nuclei in the TD when homozygous kdruq38bh mutants were compared to heterozygous and wild-type siblings (kdr+/+ embryos n = 11; kdr+/ n = 21; kdr/ n = 16). (H) There was no significant difference in the number of nuclei in ISAs or ISVs when homozygous kdruq38bh mutants were compared to wild-type siblings (n = 16, 2 ISAs or ISVs from 8 embryos). (I–K) Significant differences in LFL (I), MFL (J), and OLV (K) LEC numbers were observed when homozygous kdruq38bh mutant embryos (n = 16) were compared to kdruq38bh heterozygous (n = 22) or wild-type (n = 12) embryos. ****p < 0.0001. (L and M) In situ hybridization using probes complementary to kdr (L) and flt4 (M) in 36-hpf embryos reveals robust expression in the region where lymphatic sprouts emerged from the common cardinal vein and the primary head sinus (arrows). (N and O) Confocal images of the TgBAC(vegfd:eYFP)uq42bh; Tg(kdrl:mcherry) transgenic line at 36 hpf (N) and TgBAC(vegfd:eYFP)uq42bh at 72 hpf (O) reveal expression of vegfd in the craniofacial region close to where lymphatic sprouts emerge from the primary head sinus (arrows). (P–U) Confocal images of 5-dpf Tg(fli1a:nEGFP);Tg(5.2lyve1b:dsRed) transgenic embryos produced by in-crossing kdruq38bh/flt4hu4602 carriers. Genotypes in (P) are flt4+/+ kdr+/+, (Q) flt4+/+ kdr/, (R) flt4+/ kdr+/+, (S) flt4+/ kdr+/, (T) flt4+/ kdr/, and (U) flt4/ kdr+/+. In flt4hu4602 heterozygous embryos, loss of kdruq38bh resulted in a complete failure of the TD to form (T) and were phenotypically indistinguishable from flt4hu4602 mutant embryos. Arrows indicate TD and asterisks its absence. (V) Quantification of TD nuclei for embryos referred to in (P)–(U) (flt4+/+/kdr+/+ embryos n = 11; flt4+/+/kdr+/ n = 12; flt4+/+/kdr/ n = 4; flt4+//kdr+/+ n = 6; flt4+//kdr+/ n = 31; flt4+//kdr/ n = 16; flt4//kdr+/+ n = 8; flt4//kdr+/ n = 14; flt4//kdr/ n = 8). All scale bars in this figure represent 100 mm except for (O), in which the bar denotes 50 mm. Data are represented as the mean ± SEM.

Cell Reports 28, 2023–2036, August 20, 2019 2031

A

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Figure 5. Functional Relationship of Flt4 and Kdr In Vivo, and Summation of Ligand-Receptor Interactions (A–D) Confocal images showing facial region of 5-dpf Tg(fli1a:nEGFP);Tg(5.2lyve1b:dsRed) transgenic embryos produced by in-crossing kdruq38bh/flt4hu4602 carriers. Genotypes in (A) are flt4+/+ kdr+/+, (B) flt4+/ kdr+/, (C) flt/ kdr+/+, and (D) flt4/ kdr/. In (C) and (D), arrow indicates rostral lymph sac (RLS) and asterisk indicates its absence. All scale bars in (A)–(D) represent 100 mm. (E–G) Quantification of nuclei in lateral (LFL; E), medial (MFL; F), and otolithic (OLV; G) facial lymphatics in kdruq38bh and flt4hu4602 mutant combinations (flt4+/+/kdr+/+ embryos n = 14; flt4+/+/kdr+/ n = 13; flt4+/+/kdr/ n = 5; flt4+//kdr+/+ n = 11; flt4+//kdr+/ n = 30; flt4+//kdr/ n = 17; flt4//kdr+/+ n = 8; flt4//kdr+/ n = 19; flt4//kdr/ n = 10). (H) Quantification of RLS formation in flt4hu4602 mutant background reveals kdruq38bh mutants form fewer RLSs than kdr wild-type or heterozygotes (flt4//kdr+/+ embryos n = 8; flt4//kdr+/ n = 19; flt4//kdr/ n = 10). (I–L) Confocal images of 36-hpf Tg(fli1a:nEGFP) transgenic embryos, produced by in-crossing kdruq38bh/flt4hu4602 carriers, and immunostaining for Prox1. Merged images; green denotes EGFP-tagged Fli1-positive nuclei and red denotes Prox1. (I0 )–(L0 ) EGFP-tagged Fli1-positive nuclei only, and (I00 )–(L00 ) show Prox1 only. Yellow arrows indicate Prox1/EGFP-positive nuclei in primary head sinus (PHS); white arrows indicate common cardinal vein (CCV). Genotypes in (I) are flt4+/+ kdr+/+, (J) flt4/ kdr+/+, (K) flt4+/+ kdr/, and (L) flt4/ kdr/. All scale bars in (I)–(L) represent 50 mm and apply to all images.

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2032 Cell Reports 28, 2023–2036, August 20, 2019

et al., 1996). Zebrafish Vegfaa binds Kdr and Kdrl, as well as Flt1, but not Flt4, which is broadly consistent with human VEGFA. In contrast, zebrafish Vegfd exhibited an important difference from its human homolog in that it did not bind Flt4, demonstrated by both pull-down and surface plasmon resonance studies, whereas human VEGFD binds human VEGFR3 (Achen et al., 1998). Likewise, mouse VEGFD binds mouse VEGFR3, although it is a poor ligand for mouse VEGFR2 (Baldwin et al., 2001). Zebrafish Vegfd did bind Kdr, but not Flt1, consistent with its human homolog (Achen et al., 1998), and was unable to bind Kdrl. These data show that both Vegfc and Vegfd are capable of binding the Kdr homolog of VEGFR2, but not the Kdrl homolog, whereas Vegfaa bound both. The finding that Kdr is more similar to human VEGFR2 than Kdrl, in terms of ligand-binding specificity, is consistent with phylogenetic analysis indicating that Kdr is more closely related to VEGFR2 than Kdrl (Bussmann et al., 2007). The finding that zebrafish Vegfd does not bind Flt4 demonstrates that ligand-binding specificities of zebrafish receptors cannot be reliably inferred from those of homologous mammalian receptors and, therefore, need to be verified experimentally. Our findings that both human VEGFD and zebrafish Vegfd bind human VEGFR3, but not zebrafish Flt4, indicate there have been changes between the human and zebrafish homologs of this receptor that alter their capacity to bind Vegfd. In contrast, the capacity to bind Vegfc is not altered. From an evolutionary perspective, an ancestral Vegf receptor has been lost in eutherians (placental mammals), which have three Vegf receptors, but not in modern birds, fish, or marsupials, which have four (Bussmann et al., 2007); in zebrafish these include two receptor ohnologues (Kdr and Kdrl) that are functionally similar to human VEGFR2. We speculate that the altered selective pressure associated with these differences in the code has led to diversification of Vegf receptors due to structural alterations. For example, structural studies have identified regions of human VEGFR3 that are important for binding human VEGFC (Leppa¨nen et al., 2013), such as strand E, the C/C0 hairpin loop, and strands F, A0 , and G of the second Ig-like domain, and these regions are also likely to be important for binding human VEGFD given its close structural relationship to VEGFC (Leppa¨nen et al., 2011). Alignment of the amino acid sequences of these regions of human VEGFR3 and zebrafish Flt4 indicates multiple amino acid substitutions, deletions, and insertions (data not shown). These alterations may have resulted in functional changes, particularly in terms of VEGFD binding, but further studies are needed to define their functional significance. In terms of understanding the role of VEGFR2 family homologs in vascular development, the fact that zebrafish Kdr has taken on Vegfc- and Vegfd-mediated functions, whereas Kdrl does not bind these ligands, has created a fortuitous ‘‘conditional’’ setting. A role for VEGFR2 signaling in lymphangiogenesis has

been suggested by phenotypes in lymphatic vessels in different genetic and tissue settings in mice (Dellinger et al., 2013; Wirzenius et al., 2007), and in vitro studies showing that VEGFR2/VEGFR3 heterodimers play a role upstream of AKT and ERK signaling in LECs (Deng et al., 2015). However, current approaches in mice have been unable to segregate the observed lymphatic hypoplasia from hypoplastic angiogenesis and, as such, careful dissection of the tissue-specific roles of VEGFR2 in embryonic lymphangiogenesis remains to be reported. Here, we use zebrafish to uncover an important role for Kdr in developmental lymphangiogenesis. Kdr is essential for normal craniofacial lymphangiogenesis but not for lymphangiogenesis in the trunk although it can play a compensatory role when Flt4 dosage is reduced in the trunk. This indicates a spatially dependent role of Kdr in regulating developmental lymphangiogenesis, providing insight into organotypic modes of lymphatic development that differentially utilize VEGFR2/VEGFR3 signaling. Previous studies have shown that rostral lymphatic progenitors originate from multiple venous and non-venous origins, coalescing to form an integrated craniofacial lymphatic vasculature during zebrafish development (Astin et al., 2014; Eng et al., 2019). Additionally, the distinct signaling pathways required for craniofacial and trunk lymphangiogenesis may indicate that molecularly distinct lymphatic progenitor cells are present in different regions of the embryo as VEGFR2 and VEGFR3 signaling may elicit differential downstream pathways and even transcriptional outcomes (Deng et al., 2015). Strikingly, we show that all LEC specification in the early zebrafish embryo requires Vegfr activity. Induction of Prox1 expression in the trunk is Vegfc/Flt4-dependent (Koltowska et al., 2015; Shin et al., 2016), and, as we show here, in the face is under the control of partially compensatory Kdr and Flt4 signaling. Notably, while Prox1 is still induced in the mouse embryo upon loss of VEGFC (Karkkainen et al., 2004), the equivalent scenario as described above, where combined Kdr and Flt4 signaling induces Prox1, remains to be examined in mice. More broadly than these developmental studies, the possibility that somatic or modifier mutations in the gene for VEGFR2 in mammalian systems could contribute to diseases of the lymphatic system is an unexplored prospect. We suggest that further analysis of lymphatic phenotypes in mice with conditional ablation of the gene for VEGFR2 is warranted and that analysis of the role of VEGFR2 in lymphatic disease is a high priority. Evolution has generated remarkably diverse lymphatic vascular systems in modern vertebrates. In humans, this hierarchical network consists of initial lymphatics, pre-collecting lymphatics, collecting lymphatics, and lymphatic ducts, with distinct functional and morphological characteristics (Alitalo, 2011; Stacker et al., 2014), e.g., collecting lymphatics have specialized valves essential for unidirectional lymph flow. Teleost fishes diverged from mammals 400 million years ago and live under

(M and N) Quantification in CCV (M) and PHS (N) reveals fewer Prox1-positive nuclei in kdruq38bh/flt4hu4602 double mutants (n = 6 embryos), compared to wild-type (n = 9), flt4hu4602 (n = 8), or kdruq38bh (n = 14) mutants. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (O) Table of Vegf ligand-receptor interactions based on pull-down experiments. NT denotes not tested. (P) Schematic showing binding of zebrafish Vegf ligands to receptors, and downstream events. Mechanisms of organotypic lymphangiogenesis in the craniofacial region and trunk are indicated. mFlt1 denotes full-length transmembrane Flt1; sFlt1 soluble form; dashed arrows compensatory roles toward downstream event. Data are represented as the mean ± SEM.

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vastly different environmental conditions, such as higher ambient pressure, compared to terrestrial vertebrates so their lymphatics have divergent physical properties (Thomas and Kampmeier, 1969). For example, lymph is thought to be propelled by movement of skeletal muscle so fish have few, if any, valves and lack the hierarchy of mammalian lymphatic networks (Thomas and Kampmeier, 1969). It is not clear how this structural specialization emerged but it is possible that differences in the Vegf/Vegfr code, and altered selective pressure upon these molecular pathways, may have contributed over evolutionary time. Importantly, divergence in the Vegf/Vegfr code may also have contributed to diverse mechanisms of lymphangiogenesis, spatially, in different developing organs or tissues, thereby providing evolution the raw material needed to diversify organotypic lymphatic networks. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

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KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Cell Culture B Zebrafish Strains METHOD DETAILS B Plasmids Encoding Vegf Ligands and Soluble Receptor-Ig Fusion Proteins B Transient Transfection B Western Blotting B Receptor Binding Assays B Protein Purification B Surface Plasmon Resonance Analysis B Microinjections and Validation of Constructs In Vivo B BAC Transgenesis B Imaging B CRISPR-Genome Editing B Prox1 Immunostaining B In Situ Hybridization QUANTIFICATION AND STATISTICAL ANALYSIS B Statistical Analysis DATA AND CODE AVAILABILITY

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.07.055. ACKNOWLEDGMENTS Imaging was performed at the Australian Cancer Research Foundation’s Cancer Ultrastructure and Function Facility, Institute for Molecular Bioscience, University of Queensland. This work was supported by grants and a research fellowship from the National Health and Medical Research Council of Australia (NHMRC), and by the University of Queensland’s Centre for Cardiac and Vascular Biology and the Operational Infrastructure Program of the Victorian government. B.M.H. was supported by a National Heart Foundation/NHMRC Career Development Fellowship.

2034 Cell Reports 28, 2023–2036, August 20, 2019

AUTHOR CONTRIBUTIONS M.G.A. and B.M.H. conceived the idea and directed the work; M.G.A., B.M.H., A.J.V., N.I.B., S.P., S.A.S., M.J.G., S.J.H., S.R., and K.S.O. designed and/or performed experiments; and M.G.A., B.M.H., A.J.V., N.I.B., and S.A.S. wrote the manuscript. DECLARATION OF INTERESTS M.G.A. and S.A.S. are shareholders in Opthea, Ltd. Received: December 5, 2018 Revised: June 11, 2019 Accepted: July 16, 2019 Published: August 20, 2019 REFERENCES Achen, M.G., Jeltsch, M., Kukk, E., Ma¨kinen, T., Vitali, A., Wilks, A.F., Alitalo, K., and Stacker, S.A. (1998). Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc. Natl. Acad. Sci. USA 95, 548–553. Alitalo, K. (2011). The lymphatic vasculature in disease. Nat. Med. 17, 1371– 1380. Astin, J.W., Haggerty, M.J., Okuda, K.S., Le Guen, L., Misa, J.P., Tromp, A., Hogan, B.M., Crosier, K.E., and Crosier, P.S. (2014). Vegfd can compensate for loss of Vegfc in zebrafish facial lymphatic sprouting. Development 141, 2680–2690. Baek, S., Oh, T.G., Secker, G., Sutton, D.L., Okuda, K.S., Paterson, S., Bower, N.I., Toubia, J., Koltowska, K., Capon, S.J., et al. (2019). The alternative splicing regulator Nova2 constrains vascular Erk signaling to limit specification of the lymphatic lineage. Dev. Cell 49, 279–292.e5. Bahary, N., Goishi, K., Stuckenholz, C., Weber, G., Leblanc, J., Schafer, C.A., Berman, S.S., Klagsbrun, M., and Zon, L.I. (2007). Duplicate VegfA genes and orthologues of the KDR receptor tyrosine kinase family mediate vascular development in the zebrafish. Blood 110, 3627–3636. Baldwin, M.E., Catimel, B., Nice, E.C., Roufail, S., Hall, N.E., Stenvers, K.L., Karkkainen, M.J., Alitalo, K., Stacker, S.A., and Achen, M.G. (2001). The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man. J. Biol. Chem. 276, 19166–19171. Baldwin, M.E., Halford, M.M., Roufail, S., Williams, R.A., Hibbs, M.L., Grail, D., Kubo, H., Stacker, S.A., and Achen, M.G. (2005). Vascular endothelial growth factor D is dispensable for development of the lymphatic system. Mol. Cell. Biol. 25, 2441–2449. Bower, N.I., Koltowska, K., Pichol-Thievend, C., Virshup, I., Paterson, S., Lagendijk, A.K., Wang, W., Lindsey, B.W., Bent, S.J., Baek, S., et al. (2017a). Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish. Nat. Neurosci. 20, 774–783. Bower, N.I., Vogrin, A.J., Le Guen, L., Chen, H., Stacker, S.A., Achen, M.G., and Hogan, B.M. (2017b). Vegfd modulates both angiogenesis and lymphangiogenesis during zebrafish embryonic development. Development 144, 507–518. Bussmann, J., Bakkers, J., and Schulte-Merker, S. (2007). Early endocardial morphogenesis requires Scl/Tal1. PLoS Genet. 3, e140. Cha, Y.R., Fujita, M., Butler, M., Isogai, S., Kochhan, E., Siekmann, A.F., and Weinstein, B.M. (2012). Chemokine signaling directs trunk lymphatic network formation along the preexisting blood vasculature. Dev. Cell 22, 824–836. Covassin, L.D., Villefranc, J.A., Kacergis, M.C., Weinstein, B.M., and Lawson, N.D. (2006). Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish. Proc. Natl. Acad. Sci. USA 103, 6554–6559. Dellinger, M.T., Meadows, S.M., Wynne, K., Cleaver, O., and Brekken, R.A. (2013). Vascular endothelial growth factor receptor-2 promotes the development of the lymphatic vasculature. PLoS ONE 8, e74686.

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Petrova, T.V., and Koh, G.Y. (2018). Organ-specific lymphatic vasculature: From development to pathophysiology. J. Exp. Med. 215, 35–49.

Karkkainen, M.J., Saaristo, A., Jussila, L., Karila, K.A., Lawrence, E.C., Pajusola, K., Bueler, H., Eichmann, A., Kauppinen, R., Kettunen, M.I., et al. (2001). A model for gene therapy of human hereditary lymphedema. Proc. Natl. Acad. Sci. USA 98, 12677–12682.

Rissanen, T.T., Markkanen, J.E., Gruchala, M., Heikura, T., Puranen, A., Kettunen, M.I., Kholova´, I., Kauppinen, R.A., Achen, M.G., Stacker, S.A., et al. (2003). VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ. Res. 92, 1098–1106.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies M2-HRP antiflag mAb

Sigma-Aldrich

Cat # A8592; RRID: AB_439702

Anti-mouse IgG 800CW

LI-COR Biosciences

Cat# 925-32210; RRID: AB_2687825

Goat polyclonal anti-mouse IgG

R&D Systems

Cat # G-202-C; RRID: AB_573137

VEGFR2-Fc

R&D Systems

357-KD-050/CF

VEGFR3-Fc

R&D Systems

349-F4

NheI

New England Biolabs

R0131S

Lipofectamine 2000

Life Technologies

11668019

Flag peptide

Sigma-Aldrich

F3290-4MG

Anti-Flag affinity gel

Sigma-Aldrich

A2220-5ML

Protein A Sepharose

Sigma-Aldrich

P6649-5ML

DdeI

New England Biolabs

R0175

Anti-Prox1 antibody

Angio Bio Co

11-002

FreeStyle MAX Transfection Reagent

Life Technologies

16447100

Hi pure Plasmid Maxi prep

Life Technologies

K210007

mMessage Machine SP6 Transcription kit

Life Technologies

AM1340

Megascript SP6

Life Technologies

AM1330

RNA Clean & Concentrator

Zymoresearch

R1016

MeltDoctor HRM Master Mix

Life Technologies

4415452

In-Fusion HD Cloning Plus

Clontech

638909

European Nucleotide Archive https://www.ebi.ac.uk/ena

ENA: LR634152.1

Chemicals, Peptides, and Recombinant Proteins

Critical Commercial Assays

Deposited Data Nucleotide sequence encoding Flt1 in zFlt1(1-3)-Fc Experimental Models: Cell Lines FreeStyle 293F cells

Life Technologies

K90000-10

HEK293T

Dharmacon

#HCL4517

Zebrafish strain Tg(fli1a:EGFP)y1

Roman et al., 2002

N/A

Zebrafish strain Tg(lyve1:dsred)nz101

Okuda et al., 2012

N/A

Zebrafish strain Tg(fli1a:nEGFP)y7

Lawson and Weinstein, 2002a

N/A

This Study

N/A

pEFBOSFLAG

Achen et al., 1998

N/A

pFUSE-mIgG2Aa-Fc2 (IL2ss)

InvivoGen

# pfc2-mg2ae1

Image lab 4.1

Bio-Rad Laboratories

Version 4.1, #1709690

Zhang laboratory software

http://zlab.bio/guide-design-resources

N/A

ImageJ 1.47

National Institute of Health

N/A

Image Studio 5

LI-COR Biosciences

Version 5

Prism statistical software

Graph Pad

Version 7

Biacore S200 evaluation software

Biacore

Version 1.0

Experimental Models: Organisms/Strains

Oligonucleotides Refer to Table S1 Recombinant DNA

Software and Algorithms

Cell Reports 28, 2023–2036.e1–e4, August 20, 2019 e1

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Professor Marc Achen ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell Culture FreeStyle 293F cells were maintained in FreeStyle 293 Expression Medium (Life Technologies Australia) at 37 C in a humidified atmosphere of 8% CO2, on a shaking platform at 120 revolutions per minute (rpm). HEK293T cells were maintained in DMEM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamax and 10% FBS (Life Technologies Australia) at 37 C in a humidified atmosphere of 10% CO2. Zebrafish Strains The previously published transgenic line Tg(fli1a:EGFP)y1 (Roman et al., 2002), and a double transgenic TG(lyve1:dsred)nz101 (Okuda et al., 2012) Tg(fli1a:nEGFP)y7 (Lawson and Weinstein, 2002a) line were employed in this study. All zebrafish strains were maintained, and animal work performed, in accordance with guidelines of the Animal Ethics Committee, University of Queensland, Australia. We had no prior data to estimate our power calculation from, and so we chose the number of experiments to perform, and the parameters that we were measuring prior to performing statistical analysis. The number of experiments to perform was based on our animal ethical requirements and on analogy with previous work with zebrafish and we have presented all measurements that were taken. Embryos were randomly selected from the same tanks and embryos were collected from in-crosses of several pairs of adult zebrafish. Mutant and sibling embryos were grown together. Phenotyping preceded genotyping in mutant analyses, hence analysis was genotype blinded. Embryonic staged animals were used before sexual maturity. METHOD DETAILS Plasmids Encoding Vegf Ligands and Soluble Receptor-Ig Fusion Proteins DNA sequences for zebrafish Vegf ligands and receptors were amplified from zebrafish cDNA by PCR (the primers used are shown in Table S1). DNA encoding zebrafish Vegfaa121 (amino acids 24-138 and 183-188, lacking the signal sequence, UniProtKB: O736822), full-length zebrafish Vegfc (amino acids 20-396, lacking the signal sequence, UniProtKB: Q7T3I6) and mature Vegfc (amino acids 97-221, UniProtKB: Q7T3I6) were inserted into pEFBOSSFLAG (Achen et al., 1998) immediately downstream of DNA encoding the IL3 signal sequence and a FLAG octapeptide tag (DYKDDDDK). The encoded proteins were designated zVegfaa121-FLAG, zVegfcFULL-N-FLAG, and zVegfcDNDC-FLAG. Plasmids derived from pEFBOSSFLAG encoding a full-length form of zebrafish Vegfd tagged near the N terminus with FLAG (zVegfdFULL-N-FLAG) and a mature form of zebrafish Vegfd tagged near the N terminus with FLAG (zVegfdDNDC-FLAG), were described previously (Bower et al., 2017b). The region of Vegfd present in zVegfdDNDCFLAG is from amino acids 85-198 (UniProtKB: Q208T6). DNA encoding a longer form of mature zebrafish Vegfd, encompassing amino acids 73-198, was synthesized (Integrated DNA Technologies) and inserted into pEFBOSSFLAG – the encoded protein is N-terminally FLAG-tagged and was designated zVegfdDNDC-Long-FLAG. To generate a C-terminally FLAG-tagged form of zebrafish mature Vegfd, DNA encoding the IL3 signal sequence upstream of amino acids 85-198 of zebrafish Vegfd followed by FLAG was synthesized (Integrated DNA Technologies) and inserted into pEFBOSSFLAG that had been digested with NheI (New England Biolabs) to excise DNA encoding the IL3 signal sequence and FLAG. The C-terminally FLAG-tagged protein was designated zVegfdDNDC-FLAG-Cterm. A plasmid encoding an N-terminally FLAG-tagged version of mature human VEGFD, designated VEGFDDNDCFLAG, was described previously (Stacker et al., 1999). For all the N-terminally FLAG-tagged proteins, the two amino acids threonine followed by arginine are present between FLAG and the first amino acid of the Vegf protein. DNAs encoding the outer three Ig-like domains of zebrafish Flt1 and zebrafish Flt4 (amino acids 25-352, UniProtKB: A0A0R4IFS9), and the entire extracellular domain of Flt4 (amino acids 25-795, UniProtKB: A0A0R4IFS9), were inserted into the pFUSE-mlgG2Aa-Fc2 (IL2ss) vector (InvivoGen) between DNA for the IL2 signal sequence and the Fc region of mouse IgG (the deduced amino acid sequence for the region of Flt1 employed here is shown in Figure S4). The encoded proteins were designated zFlt1(1-3)-Fc, zFlt4(1-3)-Fc and zFlt4-Fc. Plasmids encoding protein constructs consisting of the entire regions of the extracellular domains of zebrafish Kdr and Kdrl with the Fc region of mouse IgG at the C terminus (designated zKdr-Fc and zKdrl-Fc), were described previously (Bower et al., 2017b). Transient Transfection FreeStyle 293F cells were transfected with expression vectors using FreeStyle MAX Transfection Reagent (Life Technologies Australia) according to the manufacturer. Briefly, 60 mL of cells in a 125 mL tissue culture shaker flask were transfected with 75 mL FreeStyle MAX Transfection Reagent and 75 mg of plasmid DNA in FreeStyle 293 Expression Medium. Cells were then incubated for 6 days at 37 C in a humidified atmosphere of 8% CO2 on a shaking platform at 120 rpm before collection of conditioned media. HEK293T cells were transfected using lipofectamine 2000 (Life Technologies Australia) according to the manufacturer. Briefly, cells were seeded in 150 mm tissue culture dishes and transfected with 60 mL of lipofectamine 2000 and 20 mg of plasmid DNA in e2 Cell Reports 28, 2023–2036.e1–e4, August 20, 2019

10 mL of unsupplemented DMEM. Cells were then incubated for 6 h at 37 C in a humidified atmosphere of 10% CO2 before addition of 10 mL of DMEM containing 0.4% BSA and further incubation for 24 h prior to collection of conditioned media which were clarified by centrifugation at 1500 g for 5 min at room temperature. Western Blotting Proteins were precipitated from conditioned media using Protein A-Sepharose (Sigma Aldrich) for Fc fusion proteins or anti-FLAG M2 affinity gel (Sigma-Aldrich) for FLAG-tagged proteins. Conditioned media from HEK293T cells were incubated with Protein A-Sepharose or M2 affinity gel for 3 h at 4 C, and beads collected via centrifugation (1500 g for 5 min at 4 C) and washed thrice with 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), once with 500 mM NaCl and once with 50 mM Tris-HCl (pH 8.0). Proteins were eluted from beads by incubation in reducing LDS sample buffer (Life Technologies Australia) at 95 C for 5 min, and separated by SDS-PAGE using Bolt 4%–12% Bis Tris plus gels (Invitrogen) prior to transfer to iBlot2 nitrocellulose membrane using an iBlot2 protein transfer apparatus (Life Technologies). Membranes were probed with either M2 anti-FLAG monoclonal antibody conjugated to horseradish peroxidase (HRP) (Sigma-Aldrich) for detection of FLAG-tagged proteins or anti-mouse IgG 800CW (LI-COR Biosciences) to detect Fc-tagged proteins. Detection of HRP was performed using ECL substrate (Bio-Rad Laboratories) as per manufacturer’s instructions prior to imaging on a Chemidoc gel imaging system (Bio-Rad Laboratories). Detection of the 800CW fluorophore was performed using an Odyssey CLx imaging system (LI-COR Biosciences). All western blots shown in figures were conducted at least three times, as part of independent experiments assessing proteolytic processing of proteins or receptor binding by ligands, with the same results. Receptor Binding Assays Soluble Vegf receptor Fc-fusion proteins were precipitated from conditioned media by incubation with Protein A-Sepharose (Sigma Aldrich) for 3 h at 4 C followed by centrifugation (1500 g for 5 min at 4 C). The resulting beads were then incubated with conditioned media containing FLAG-tagged Vegf ligands for 16 h at 4 C. Alternatively, 1 mg of human VEGFR2-Fc or VEGFR3-Fc fusion proteins (R&D Systems) were incubated with Protein A-Sepharose prior to centrifugation and incubation with conditioned media containing FLAG-tagged Vegf ligands, as above. Beads were collected by centrifugation (1500 g for 5 min at 4 C) and washed thrice with 0.1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), once with 500 mM NaCl and once with 50 mM Tris-HCl (pH 8.0). Proteins were then eluted by incubation in reducing LDS sample buffer (Life Technologies Australia) at 95 C for 5 min and analyzed by SDS-PAGE/ Western blotting as described above. Prior to all pull-down experiments, the presence of FLAG-tagged Vegf ligands in samples of conditioned media was confirmed by precipitation and western blotting with anti-FLAG antibodies. Negative controls for receptor binding assays involved: (i) Use of the Fc of mouse IgG in ligand pull-downs, instead of soluble Vegf receptors, to ensure there were no ‘‘non-specific’’ interactions between Fc in the soluble receptors and Vegf ligands; (ii) Use of conditioned media derived from cells transfected with expression vector lacking DNA encoding any Vegf ligand to demonstrate that signals detected from conditioned media containing recombinant Vegf ligands arose from these ligands rather than other components of the media. Measures taken to ensure appropriate and matched input levels of ligands and of receptors were as follows: (i) Positive pull-downs for each Vegf ligand were included in which the ligand was precipitated by a soluble Vegf receptor and successfully detected by western blot. The same volume of the conditioned medium containing a given Vegf ligand was used for every pull-down involving the ligand, ensuring a matched input level of that ligand which was sufficient to allow detection of it by western blot if a soluble receptor could bind it; (ii) Positive pull-downs were conducted in which each soluble Vegf receptor precipitated a Vegf ligand that was then successfully detected by western blot. The same amount of a given soluble receptor was used in all pull-downs involving the receptor, ensuring that a sufficient and matched input level of that receptor was employed. All receptor binding experiments were conducted at least three times, using fresh conditioned media, with the same results. Protein Purification Proteins were purified from conditioned media of transiently transfected FreeStyle 293F cells by affinity chromatography using antiFLAG M2 affinity gel (Sigma-Aldrich) for FLAG-tagged proteins or Protein A-Sepharose beads for Fc-tagged proteins. Briefly, 30 mL of conditioned media was added to 1 mL of beads and incubated at 4 C for 3 h. Beads were then washed with 20 mM Tris.HCl, 150 mM NaCl, pH 7.6 before elution with 100 mg/ml FLAG peptide (Sigma-Aldrich) for FLAG-tagged proteins or 100 mM glycine pH 2.8 for Fc-tagged proteins. The FLAG peptide was removed and buffers exchanged to PBS using centrifugal concentrators (Amicon). Surface Plasmon Resonance Analysis Binding kinetics were analyzed using a BIAcore S200 optical biosensor (GE Healthcare). Goat polyclonal anti-mouse IgG (R&D Systems) (0.1 mg/ml in 10 mM acetate, pH 4.5) was immobilized on a CM5 chip using amine coupling with 10 mM HEPES, 150 mM NaCl, 0.005% Tween-20, pH 7.4, as running buffer at 25 C. Immobilization levels of 12822 RU-15016 RU were achieved. Prior to protein capture, high affinity antibody-binding sites refractory to regeneration were blocked by injection of Fc at 0.7 mM followed by regeneration with 0.1 M phosphoric acid. zFlt4-Fc or Fc control were captured at the start of every cycle by injection for 120 s at a concentration of 0.7 mM (with capture levels of 683-734 RU for Fc and 559-766 RU for zFlt4-Fc) and regenerated at the end of every cycle with injection of 0.1 M phosphoric acid for 30 s. Using a single-cycle kinetic method zVegfaa121-FLAG, zVegfcDNDC-FLAG or zVegfdDNDC-FLAG, were injected for 240 s at 6 different concentrations in a 3-fold dilution series, from 3.6 nM to 878 nM or

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4.1 nM to 1000 nM, at a flow rate of 30 ml/min and a dissociation time of 600-3600 s. Binding experiments were performed at 25 C with 1 mg/ml CM dextran, 0.05% Tween-20 in PBS, pH 7.4 as running buffer. Data were analyzed using Biacore S200 evaluation software version 1.0, and fitted to a 1:1 binding model. Microinjections and Validation of Constructs In Vivo The previously published transgenic line Tg(fli1a:EGFP)y1 was used for mRNA injections and imaging (Roman et al., 2002), while a double transgenic TG(lyve1:dsred)nz101 (Okuda et al., 2012) Tg(fli1a:nEGFP)y7 (Lawson and Weinstein, 2002a) line was used for CRISPR genome editing. DNA encoding receptor constructs, including the interleukin-2 (IL2) secretion signal and the Fc domain, were amplified and cloned into the pCS2+ vector using Infusion enzyme (Clontech, CA, USA), and thereafter, capped mRNAs were transcribed and 250 pg injected into the yolk of 1-cell stage embryos as described (Hogan et al., 2009b). Full-length forms of Vegfc or Vegfd (derived from previously described cDNA clones (Hogan et al., 2009a)) were used to generate vegfc or vegfd mRNA for injections. Receptor-Fc constructs were validated in zebrafish embryos by mRNA injection: zFlt4(1-3)-Fc and zFlt4-Fc blocked secondary angiogenesis, a Vegfc-dependent process, as expected (Figure S1B,D&E) and soluble Flt1 and zKdrl-Fc reduced or blocked primary angiogenesis, a Vegfaa-dependent process, as expected (Figures S1A and S1C). Injection of mRNA for zKdr-Fc did not give phenotypes which could have been due to this construct not being sufficiently expressed in vivo, so we addressed the role of Kdr by generation of a CRISPR mutant. BAC Transgenesis The vegfd:eYFP BAC transgenic line was generated as previously described (Hogan et al., 2009a) using the CH73-374B19 clone and the homology-tagged primers listed in Key Resources Table. TgBAC(vegfd:eYFP)uq42bh transgenic carrier adults were identified based on expression of eYFP and confirmed by sequencing. Imaging Zebrafish embryos were mounted in 0.5% low-melting agarose and imaged using a Zeiss LSM 710 FCS confocal microscope. Images were processed using ImageJ 1.47 software (National Institutes of Health). For facial lymphatic quantification, LECs were manually counted using ImageJ. Quantification of bilateral turning of arterial intersegmental vessels at 28-32 hpf, and of parachordal LECs (PLs) at 54 hpf was performed by live scoring using a Leica M165FC microscope. CRISPR-Genome Editing CRISPR-genome editing of kdr was performed as previously described (Gagnon et al., 2014). The guide RNA was generated using the Zhang laboratory software (http://zlab.bio/guide-design-resources), transcribed with Ambion Megascript SP6 promoter kit (Ambion) and purified using the RNA Clean & ConcentratorTM (Zymoresearch). One mg of Cas9 mRNA and 300 pg of guide RNA were injected into single-cell stage embryos. Fish harboring mutations were identified by HRMA (Key Resources Table), and the 2-bp deletion mutants genotyped by DdeI restriction digest of PCR product amplified with primers listed in Key Resources Table. Prox1 Immunostaining Prox1 immunostaining was performed as previously described (Okuda et al., 2018). In Situ Hybridization In situ hybridization was performed as previously described (Hogan et al., 2009b). QUANTIFICATION AND STATISTICAL ANALYSIS Statistical Analysis Statistical analysis was performed using Prism software (GraphPad). When data conformed to parametric assumptions, one-way ANOVA using Fisher’s individual error post hoc test was used to identify significant differences. ANOVA F values followed by degrees of freedom (in brackets) are as follows: for Figure 2B and 9.411 (6, 294); Figure 2D, 196 (2, 88); Figure 3E, 224.9 (2, 82); Figure 4G and 2.12 (2, 45); Figure 4H, 37.36 (2, 47); Figure 4I, 27.15 (2, 45); Figure 4J, 43.47 (2, 46); Figure 4V, 41.47 (8, 102); Figure 5E, 35.87 (8, 118); Figure 5F, 32.56 (8, 118); Figure 5G, 38.67 (8, 115); Figure 5M and 7.538 (3, 33); Figure 5N, 14.47 (3, 33); Figure S1C, 510.2 (4, 156); Figure S1D, 208.4 (4, 134). Statistical details of experiments can be found in figure legends. All data are represented by the mean, and error bars represent standard error of the mean. DATA AND CODE AVAILABILITY The accession number for the region of zebrafish Flt1 included in the zFlt1(1-3)-Fc construct reported in this paper is ENA: LR634152.1.

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