Ccbe1 Regulates Vegfc-Mediated Induction of Vegfr3 Signaling During Embryonic Lymphangiogenesis Ludovic Le Guen1,*, Terhi Karpanen2, Dörte Schulte2, Nicole C

RESEARCH ARTICLE

Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis Ludovic Le Guen1,*, Terhi Karpanen2, Dörte Schulte2, Nicole C. Harris3,4, Katarzyna Koltowska1, Guy Roukens2, Neil I. Bower1, Andreas van Impel2, Steven A. Stacker3,4, Marc G. Achen3,4, Stefan Schulte-Merker2,5,‡ and Benjamin M. Hogan1,‡,§

ABSTRACT lineage, and express Prox1, but fail to migrate from the cardinal vein The VEGFC/VEGFR3 signaling pathway is essential for (Karkkainen et al., 2004; Hägerling et al., 2013). Reduced lymphangiogenesis (the formation of lymphatic vessels from pre- development, or impaired function of lymphatics, leads to tissue fluid existing vasculature) during embryonic development, tissue accumulation and lymphedema in humans, which can be inherited as regeneration and tumor progression. The recently identified secreted a result of mutations in key developmental genes (Karkkainen et al., protein CCBE1 is indispensible for lymphangiogenesis during 2000; Irrthum et al., 2003; Alders et al., 2009; Connell et al., 2010). development. The role of CCBE1 orthologs is highly conserved in A mutation in VEGFC has recently been shown to be responsible for zebrafish, mice and humans with mutations in CCBE1 causing inherited lymphedema (Gordon et al., 2013). generalized lymphatic dysplasia and lymphedema (Hennekam Vegfr3-deficient mice die early during gestation (embryonic day syndrome). To date, the mechanism by which CCBE1 acts remains 9.5) owing to cardiovascular failure (Dumont et al., 1998), unknown. Here, we find that ccbe1 genetically interacts with both consistent with the expression of Vegfr3 in blood vascular vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by endothelium in the early embryo. However, after midgestation, increased Vegfc are suppressed in the absence of Ccbe1, and Vegfc- Vegfr3 expression becomes enriched in the developing lymphatic driven sprouting is enhanced by local Ccbe1 overexpression. vasculature (Kaipainen et al., 1995) and Vegfr3 signaling is Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in sufficient to initiate lymphangiogenesis (Veikkola et al., 2001). the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the Importantly, patients with mutations in VEGFR3 (FLT4 – Human levels of fully processed, mature VEGFC in vitro and the Gene Nomenclature Database) develop Milroy’s disease, overexpression of mature VEGFC rescues ccbe1 loss-of-function characterized by reduced lymphatic drainage and lymphedema in the phenotypes in zebrafish. Taken together, these data identify Ccbe1 lower limbs (Karkkainen et al., 2000). These studies, and many as a crucial component of the Vegfc/Vegfr3 pathway in the embryo. more, have shown that the precise modulation of the Vegfc/Vegfr3 signaling pathway is crucial during lymphatic vascular development. KEY WORDS: Angiogenesis, Lymphangiogenesis, Lymphedema, In zebrafish, lymphatic vascular development initiates from Vasculature, Zebrafish 32 hours post-fertilization (hpf), when precursor cells first migrate dorsally from the cardinal vein to colonize the horizontal INTRODUCTION myoseptum, generating a population of parachordal The lymphatic vasculature develops from pre-existing vessels in a lymphangioblasts (PLs) (Küchler et al., 2006; Yaniv et al., 2006; dynamic process called lymphangiogenesis, and is necessary to Hogan et al., 2009a; Isogai et al., 2009). These PLs subsequently preserve tissue fluid homeostasis, for fat absorption and normal migrate alongside arteries, both dorsally and ventrally (from ~60 immune function. The lymphatic vascular network originates in the hpf), and remodel into the major trunk lymphatic vessels (Bussmann mouse embryo from the cardinal vein, where lymphatic endothelial et al., 2010; Cha et al., 2012), forming the thoracic duct, precursor cells first bud and migrate dorsolaterally away from the vein intersegmental lymphatic vessels and dorsal longitudinal lymphatic under the control of Vegfc and its receptor Vegfr3 (Flt4 – Mouse vessels (for reviews, see Koltowska et al., 2013; van Impel and Genome Informatics). In mice, the overexpression of Vegfc in the skin Schulte-Merker, 2014). In addition, a complex craniofacial results in dramatic hyperplasia of lymphatic vessels (Jeltsch et al., lymphatic network is formed (Okuda et al., 2012). Zebrafish vegfr3 1997), whereas Vegfc knockout mice develop lymphatic hypoplasia (flt4 – Zebrafish Information Network) and vegfc function is and lymphedema (Karkkainen et al., 2004). In developing Vegfc essential for all secondary (venous) angiogenesis, including the knockout embryos, endothelial cells are still specified to the lymphatic sprouting of lymphatic precursors from the cardinal vein and subsequent formation of PLs (Isogai et al., 2003; Küchler et al., 1Division of Molecular Genetics and Development, Institute for Molecular 2006; Yaniv et al., 2006; Hogan et al., 2009a). Bioscience, The University of Queensland, St Lucia, QLD 4073, Australia. We have previously reported two zebrafish mutants with an 2 Hubrecht Institute – KNAW & UMC Utrecht, 3584 CT Utrecht, The Netherlands. absence of lymphatic (but not blood vascular) development (Hogan 3Tumour Angiogenesis Programme, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, VIC 8006, Australia. 4Sir Peter MacCallum et al., 2009a; Hogan et al., 2009b). These phenotypes were caused Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia. by mutations in vegfr3 and the previously uncharacterized gene 5 EZO, WUR, 6708LX Wageningen, The Netherlands. collagen and calcium-binding EGF domains-1 (ccbe1) (Hogan et *Present address: U1046, Université Montpellier 1, Université Montpellier 2, 34967 CEDEX 2 Montpellier, France. al., 2009a; Hogan et al., 2009b). ccbe1 was shown to act at stages ‡These authors contributed equally to this work identical to vegfr3 and vegfc (Hogan et al., 2009a). ccbe1 encodes an extracellular matrix (ECM) protein, and is composed of N- §Author for correspondence ([email protected]) terminal calcium-binding epidermal growth factor (EGF)-like and

Received 26 June 2013; Accepted 30 December 2013 EGF domains, and two collagen-repeat domains towards the C Development

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Fig. 1. See next page for legend. Development

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Fig. 1. Zebrafish vegfc mutants lack lymphatic vessels, but appear from vegfr3 and ccbe1 mutants at the level of gross embryo otherwise normal. (A-D) Confocal projections of Tg(fli1a:EGFP) show morphology (supplementary material Fig. S1A-D), and that unaltered overall morphology and blood vasculature in vegfchu5055 mutants displayed a severe reduction in secondary angiogenesis and block in hu5055 (B) compared with wild type (A). The TD (C, arrows) is absent in vegfc lymphatic precursor sprouting from the cardinal vein (Fig. 1A-D; (D, asterisks). (E) Positional cloning linked alleles hu6124 and hu5055 to large regions of chromosome 1, containing the vegfc gene. For hu6124, 13 Fig. 2A-H; supplementary material Fig. S1E-K). At 5 days post- recombinant embryos identified at z9394 were reduced to four recombinant fertilization (dpf), the hu5055, hu6124, hu5142 and hu6410 mutants embryos common with z5508, identifying linkage in a large region containing have a grossly normal, functional blood vasculature, but the vegfc. For hu5055, eight recombinant embryos at z2691 and 33 different lymphatic vasculature is absent (Fig. 1B,D). Using a positional recombinant embryos at z4694 flanked a large region of chromosome 1, also cloning approach, we found that these mutants were linked to containing vegfc. (F) Sequencing of vegfc alleles. hu6410 encodes an early chromosome 1, containing the vegfc gene (Fig. 1E; data not shown). stop allele leading to a predicted truncated protein lacking the core (VHD) Sequencing of vegfc in hu5055 mutants revealed a missense region. hu5142, hu6124 and hu5055 encode missense mutations, all in highly conserved regions of the protein (G) and predicted to be damaging by mutation changing a cysteine into an arginine residue (C365R) in PolyPhen-2 (Adzhubei et al., 2010) with scores of 1 out of 1. (G) Alleles the highly conserved C-terminal propeptide (Fig. 1F,G). Additional hu5055, hu6410, hu6124 and hu5142 all encode mutations in regions that mutations identified caused a premature stop in hu6410 (L107X), a are conserved across multiple species. (H) Alleles hu5055 and hu6410 fail to phenylalanine to cysteine change in the highly conserved VHD- complement, with trans-heterozygote embryos displaying a lack of lymphatic VEGF homology domain (F138C) and another cysteine-to-serine structures; phenotype scored as percentage extent of thoracic duct over ten substitution in the C-terminal propeptide of Vegfc (C339S) somites (total number of embryos scored n=212). (I,J) The penetrance of vegfc mutant phenotypes varies, as shown by the variable number of PLs (I) (Fig. 1F,G). All mutations were predicted to be damaging by or TD extent (J). The hu6410 allele (L107X) shows the most severe defects PolyPhen-2 (Adzhubei et al., 2010). These vegfc mutations in heterozygous and homozygous mutants, whereas hu5055 does not show segregate with the thoracic duct (TD) deficiency phenotype and haplo-insufficiency phenotypes. (K) vegfchu5055 genetically interacts with phenocopy MO-vegfc knockdown or soluble Vegfr3 ligand trap- vegfr3hu4602 in trans-heterozygous embryos. Confocal projections of induced phenotypes (Küchler et al., 2006; Yaniv et al., 2006; Hogan Tg(fli1a:EGFP; kdr-l:mCherry) showing examples of the heterogeneity et al., 2009a; Hogan et al., 2009b). Furthermore, these phenotypes observed during TD development at 5 dpf in vegfc+/−; vegfr3+/− embryos. Arrows indicate the thoracic duct, asterisks indicate absence of thoracic duct. are consistent with recently described phenotypes in a Vegfc truncation mutant (Villefranc et al., 2013). Interestingly, scoring both TD extent and PL number, we noted terminus of the protein. The protein localizes to secretory vesicles variable penetrance of different alleles and haplo-insufficient (Alders et al., 2009), and is secreted to the ECM, where it binds phenotypes for the more penetrant alleles (Fig. 1I,J). The hu5055 proteins such as collagens or vitronectin (Bos et al., 2011). allele segregated in a Mendelian, autosomal recessive manner and was Consistently, Ccbe1 functions non-cell-autonomously in zebrafish thus used in subsequent assays. Crossing hu5055 mutant carriers to (Hogan et al., 2009a). There are no described receptors or pathways the previously described expando/vegfr3 mutant (Hogan et al., 2009b), known to interact with Ccbe1, with progress limited at least in part we found an increased frequency of TD defects consistent with by the fact that full-length CCBE1 protein is insoluble in many genetic interaction in the same pathway (Fig. 1K; Fig. 2I). standard assays (S.S.-M. and B.M.H., unpublished observations). Importantly, mice deficient for Ccbe1 lack lymphatic vasculature ccbe1, vegfr3 and vegfc zebrafish mutants genetically (Bos et al., 2011) and mutations in human CCBE1 are causative for interact generalized lymphedema and lymphangiectasia in Hennekam The ccbe1hu3613, vegfr3hu4602 and vegfchu5055 mutants display a syndrome (Alders et al., 2009; Connell et al., 2010). Interestingly, complete loss of the lymphatic vasculature when homozygous treatment with Ccbe1 protein (a truncated soluble form) increased (Fig. 2A-H). Previous, non-quantitative observations (Hogan et al., Vegfc-induced lymphangiogenesis in a cornea micropocket assay (Bos 2009a; Hogan et al., 2009b) indicated that partial loss of ccbe1 or et al., 2011). Furthermore, mice heterozygous for both Vegfc and vegfr3 led to phenotypically wild-type lymphatic development. We Ccbe1 show a genetic interaction in lymphatic development took advantage of these mutants to determine more rigorously if (Hägerling et al., 2013). These results were suggestive that Ccbe1 and combined heterozygous mutations led to enhanced lymphatic Vegfc can function together in lymphangiogenesis, but mechanistic phenotypes. We crossed heterozygous carriers and quantified the insights into the molecular function of Ccbe1 have remained elusive. extent of the TD across ten body segments in the trunks of Here, we have investigated the relationship between Ccbe1 and the individual embryos. Subsequent genotyping first confirmed the Vegfc/Vegfr3 signaling pathways. Using genetic epistasis and analysis genetic interaction in vegfchu5055/+, vegfr3hu4602/+ double of signaling events in vivo, we show that the embryo requires Ccbe1 heterozygous animals (Fig. 2I). We found that ~28% of scored for normal Vegfc/Vegfr3/Erk signaling. CCBE1 is capable of embryos displayed ≤50% TD extent; within this population, 71% of increasing the levels of mature, processed VEGFC in vitro, which can the embryos were double heterozygotes for vegfc and vegfr3, a rescue ccbe1 loss-of-function phenotypes when overexpressed in vivo. significant enrichment from total double heterozygosity of 27% Together, our findings suggest that Ccbe1 activates Vegfc through its (Fig. 2I). Importantly, similar robust interactions of vegfr3 and vegfc processing and release from the ECM in order to regulate were observed with ccbe1. The population of embryos displaying lymphangiogenesis and venous sprouting in the embryo. These ≤50% TD extent from a ccbe1hu3613/+, vegfr3hu4602/+ cross was findings suggest common mechanisms in development and vascular enriched (54%) for double heterozygotes, as were embryos from a pathologies (e.g. lymphedema) and further suggest Ccbe1 as a new ccbe1hu3613/+, vegfchu5055/+ cross (61%) (Fig. 2J,K). This genetic therapeutic entry point in treating pathological lymphangiogenesis. interaction seems to be selective to the ccbe1hu3613, vegfr3hu4602 and vegfchu5055 mutants because in crosses to two other, as yet genetically RESULTS uncharacterized lymphatic mutants, we did not observe any Identification of four zebrafish vegfc mutant alleles interaction (L.L.G. and B.M.H., unpublished observations). Finally, In a forward genetic screen for zebrafish lymphatic vascular we generated triple heterozygote embryos and found that the

mutants, we identified several mutants that were indistinguishable population developing ≤50% TD extent contained most of the triple Development

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Fig. 2. ccbe1, vegfc and vegfr3 genetically interact in double and triple heterozygous animals. (A-H) Confocal projections of Tg(fli1a:EGFP; kdr- l:mCherry) show grossly unaltered overall morphology and blood vasculature in ccbe1hu3613 (B), vegfr3hu4602 (E) and vegfchu5055 (F) mutants compared with wild type (A). The TD (C, arrows) is absent in ccbe1hu3613 (D), vegfr3 hu4602 (G) and vegfc hu5055 (H) mutants (asterisks). (I-K) ccbe1, vegfc and vegfr3 genetically interact in double heterozygote embryos, which display lymphatic defects. Offspring from vegfc+/− and vegfr3+/− carriers give rise to 28% of embryos (n=28/99) with a TD length of ≤50%. This population is significantly enriched (71%; n=20/28; P<0.0001) in double heterozygotes (I). Similarly in ccbe1+/− and vegfr3+/− crosses, 24% (n=24/100) embryos develop ≤50% of their TD, and again this population is significantly enriched (54%; n=13/24; P=0.0098) in double heterozygotes (J). In ccbe1+/− and vegfc+/− crosses, 14% (n=18/126) of embryos develop ≤50% of their TD, this population being significantly enriched (61%; n=11/18; P=0.0096) in double heterozygotes (K). (L) Crossing double heterozygous ccbe1+/−; vegfr3+/− animals to vegfc+/− animals, results in 21% (n=25/121) of embryos with ≤50% of TD: within this population, 40% (n=10/25; P=0.0004) of the embryos were triple heterozygotes, a significant enrichment from the statistical triple heterozygosity rate of 15%. heterozygote embryos, demonstrating a strong interaction (Fig. 2L). 2007; Hogan et al., 2009b). We took advantage of the hyperbranching This synergistic genetic interaction shows that lymphatic phenotype to investigate ccbe1 function. In clutches of embryos development is sensitive to the level of activation of the Vegfc/ derived from incrosses of ccbe1 heterozygous carriers, we activated Vegfr3 pathway, and that altering ccbe1 dosage modifies phenotypes the Vegfc/Vegfr3 pathway in arteries by knocking down dll4. in this pathway. Combined with the fact that ccbe1, vegfc and vegfr3 Embryos were first sorted at 72 hpf according to the severity of act at the same stage during the cellular progression of arterial hyperbranching, and then subsequently genotyped. The lymphangiogenesis and that these mutants present the most selective population containing animals with vastly reduced or no loss of lymphatic vascular structures that we have observed, we hyperbranching (Fig. 3Aiii, graph) was significantly enriched in considered this a strong indication that Ccbe1 could be a novel ccbe1hu3613 mutants (81%; P<0.0001) whereas the category containing component of the Vegfc/Vegfr3 signaling pathway. severe aISV hyperbranching (Fig. 3Aii) was enriched in wild-type and heterozygote embryos (83%; P=0.0019; Fig. 3A, graph). Hence, ccbe1 mutation suppresses phenotypes driven by ectopic ccbe1 mutation suppressed the dll4 loss-of-function phenotype. Vegfc/Vegfr3 signaling in embryonic arteries To validate this observation further, we used a second phenotypic Previous studies have shown that the knockdown of dll4 leads to an assay that has been previously described (Hogan et al., 2009b). When excessive intersegmental artery (aISV) angiogenesis phenotype by 72 vegfc is overexpressed by mRNA injection and dll4 is simultaneously hpf (Leslie et al., 2007; Siekmann and Lawson, 2007; Hogan et al., depleted, we observe ectopic turning of aISVs in the trunk from as 2009b). This phenotype occurs because in wild-type arteries, the early as 30 hpf (compare Fig. 3Bi with 3Bii) that is not seen in single Vegfc/Vegfr3 pathway is inhibited by Dll4. Depletion of Dll4 leads to treatment controls (supplementary material Fig. S2). aISVs deficient increased activation of Vegfc/Vegfr3 signaling in aISVs, resulting in for dll4 are more responsive to vegfc RNA injection (Hogan et al.,

aISV hyperbranching (Leslie et al., 2007; Siekmann and Lawson, 2009b) and phenotypes appear the same as those observed when vegfc Development

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Fig. 3. Phenotypes driven by ectopic Vegfc/Vegfr3 signaling are suppressed in ccbe1-deficient embryos. (A) At 72 hpf, dll4 morphants display an arterial hyperbranching phenotype (arrow) driven by increased Vegfc/Vegfr3 signaling in the transgenic Tg(fli1a:EGFP) line. This phenotype was suppressed in ccbe1hu3613 mutants. Eighty-one per cent of MO-dll4 injected embryos displaying wild-type or mild phenotypes were ccbe1 mutants (n=17/21), whereas the population displaying the most severe phenotype was mainly composed of wild-type or heterozygous siblings (83%; n=139/168). (B) In dll4 morphants, arteries are sensitized to increased vegfc expression during primary sprouting. Arteries in MO-dll4, vegfc mRNA-injected embryos display aberrant, ectopic turning (arrow) as early as 30 hpf. Embryos from ccbe1 carrier incrosses, injected with 100 ng vegfc mRNA and 5 ng MO-dll4, were sorted into the phenotypic categories ‘wild type’ and ‘severe’. Genotyping revealed that 70% of the embryos displaying wild-type morphology were ccbe1 mutants (n=19/27). By contrast, the population affected by the most severe phenotype was composed of 83% wild-type or heterozygous siblings (n=122/147). (C) Confocal projections of Tg(fli1a:EGFP; flt1:tomato; hsp70l:Gal;4XUAS:vegfc) embryos show that endothelial cells in heat-shocked embryos display aberrant ectopic branching at 72 hpf. The ectopic endothelial cells are venous derived (flt1:tomato negative, arrow in Ciii). Heat-shocked embryos that were injected with 2.5 ng of MO-ccbe1 do not show this phenotype (asterisks). Scoring of the number of aberrant vISVs per heat-shocked embryo showed a significant rescue (0.12 in MO-ccbe1 injected n=22, versus 4.76 in uninjected controls n=25; P<0.0001) of the phenotype. is expressed from the hsp70l promoter (Nicoli et al., 2012). In clutches suppressed in ccbe1hu3613 mutants, and additionally that a Vegfc- and of embryos derived from ccbe1 heterozygous incrosses, we Vegfr3-driven venous phenotype is rescued in ccbe1 morphant ectopically activated the Vegfc/Vegfr3 pathway by injecting vegfc embryos. These findings suggest that Ccbe1 is necessary for the mRNA and a dll4 morpholino. Animals were sorted at 30 hpf function of the Vegfc/Vegfr3 signaling pathway during embryonic according to the severity of aISV phenotype, and then genotyped. Of development. the embryos displaying a weak phenotype, or no phenotype at all (Fig. 3Biii), 70% were ccbe1 mutants (Fig. 3B, graph), a highly Vegfr3-dependent Erk activation in embryonic veins significant (P<0.0001) enrichment. The population displaying severe requires ccbe1 phenotypes was significantly enriched in wild-type and heterozygote It is well established that VEGFR3 signals via intracellular kinases animals (83%; P=0.0006; Fig. 3B, graph). Hence, ccbe1 mutation that include ERK (reviewed by Bahram and Claesson-Welsh, 2010). suppressed the dll4 loss-of-function, vegfc overexpression phenotype. We decided to further investigate downstream signaling pathways to determine if Ccbe1-deficient embryos display a signaling block Ccbe1 knockdown suppresses phenotypes driven by ectopic in venous endothelium. We used whole-mount immunofluorescence Vegfc/Vegfr3 signaling in embryonic veins to examine phospho-Erk in the context of the embryonic We next generated a new zebrafish transgenic line vasculature. The signal observed was specific as it was reduced in (hsp70l:GAL4;UAS:vegfc), which ubiquitously expresses full-length embryos treated with the MEK inhibitor PD98059 (which inhibits vegfc under the control of the heat shock (hsp70l) promoter Mek activation and hence Erk phosphorylation) and was ectopically (supplementary material Fig. S3A,B) (Scheer et al., 2001). In these induced in the ventral posterior cardinal vein (PCV) in the context embryos (following two consecutive heat shocks at 28 and 56 hpf), of vegfc overexpression (Fig. 4A). In wild-type embryos, we found veins sprout ectopically, resulting in hyperbranched intersegmental that Erk activity was broadly detected in the neural tube, muscle and vessels (vISVs) as visualized in fli1a:GFP, flt1:tomato double epithelia, but also in the endothelium of the PCV. In PCV transgenic animals (Fig. 3Ciii). ccbe1 morpholino injection rescued endothelium, Erk activation was segmented and dorsally enriched this phenotype, blocking excessive venous sprouting (Fig. 3Civ, in individual cells at 32 hpf, concomitant with the induction of graph; P<0.0001). secondary sprouting. Importantly, this patterned activation of Erk Taken together, we found in two blind, genotype-based was markedly reduced in both MO-vegfr3- and MO-ccbe1-injected

experiments, that Vegfc- and Vegfr3-driven arterial phenotypes are embryos compared with wild-type controls (Fig. 4B,C). These Development

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Fig. 4. Vegfr3-dependent Erk signaling requires ccbe1 during the induction of secondary sprouting in zebrafish. (A) Analysis of phospho-Erk (P-Erk) expression in 32 hpf embryos. P-Erk (green) and fli1a:EGFP (white) images (lateral view) show P-Erk detected broadly in whole-mount and cross-sectioned (right-hand panels, merge upper, P-Erk lower) control embryos. Signal was increased in Vegfc-induced (dll4 MO + vegfc mRNA-injected) embryos in the posterior cardinal vein (n=8/8; Vegfc-induced embryos all showed ectopic expression in the ventral wall of the PCV). Cross section merged channel images shown in a and b, P-Erk only in c and d. Treatment with the Erk inhibitor PD98059 led to a reduction in all P-Erk staining. Arrows indicate P-Erk expression in the dorsal PCV. DA (dorsal aorta) and PCV (posterior cardinal vein) are indicated. (B) Comparison of P-Erk staining in control uninjected (left), MO-vegfr3 and MO-ccbe1 embryos. Upper panels are merged images and lower P-Erk only, viewed laterally (left) and cross-sectioned (right). Cross sections (right) are from separate embryos. Arrows indicate P-Erk expression in the dorsal PCV. DA and PCV are indicated. (C) Quantification of P-Erk-positive cells in the cardinal vein located in the dorsal compared with ventral wall (left-hand graph). Scores through individual sections of z-stack images from 12 control embryos, scored laterally across three somites in the trunk. Quantification of P-Erk-positive cells in the cardinal vein in control and MO-injected conditions (right-hand graph) (scores from n=10 control embryos, n=13 MO-vegfr3-injected and n=15 MO-ccbe1-injected embryos). (D) Immunoprecipitation (IP) and western blot (IB) detection of phosphorylated Vegfr3 at 32 hpf in wild type and in ccbe1, vegfr3, vegfc morphant and vegfc mRNA-injected embryos. The level of phosphorylated Vegfr3 is markedly reduced in ccbe1, vegfr3 and vegfc morphants, but is increased in vegfc-mRNA injected (500 ng) embryos compared with wild type (D, upper blot, IP for phospho-Vegfr3 and IB detection with phospho-Vegfr3). Loading controls were: the IgG light chain [IgG(l)] present in all blots after IP (D, middle blot), and Myosin to monitor protein input in IPs (D, lower blot). Quantification of Vegfr3 phosphorylation (relative to the loading control) based on three independent experiments is shown in right-hand panel. The decrease in MO-ccbe1 compared with uninjected controls is statistically significant (P<0.05). (E) qPCR analysis of the expression of ccbe1, vegfr3, vegfc, kdr and kdrl in uninjected control and MO-ccbe1-, MO-vegfc-, and MO-vegfr3-injected embryos. Error bars represent s.d. (C) or s.e.m. (D,E). findings confirm the presence of Vegfc- and Vegfr3-dependent Erk zebrafish Vegfr3 are unavailable, but we found two commercial signaling in embryonic veins and demonstrate that Ccbe1 is antibodies directed against conserved regions of human VEGFR3 necessary for activation of this pathway. that cross-reacted with zebrafish Vegfr3. One of these was directed We also examined the activation status of Vegfr3 by western blot against the phosphorylated residues Tyr1063/1068 (human) or of embryonic lysates. The phosphorylation of Tyr1063/1068 in Tyr1071/1076 in zebrafish (supplementary material Fig. S4). Using human VEGFR3 reflects the activation status of VEGFR3, this phospho-VEGFR3 antibody for western blotting we detected promoting downstream signaling essential for lymphangiogenesis Vegfr3 in zebrafish lysates immunoprecipitated with the same

(Dixelius et al., 2003; Salameh et al., 2005). Antibodies against antibody at 32 hpf, during initiation of secondary sprouting. The Development

1244 RESEARCH ARTICLE Development (2014) doi:10.1242/dev.100495 signal was increased in vegfc mRNA-injected embryos, but was At 48 hpf, ccbe1-overexpressing animals still show no phenotype, reduced in MO-vegfr3- and MO-vegfc-injected embryos. A similar whereas vegfc-overexpressing animals display vastly increased reduction in phospho-Vegfr3 was observed in MO-ccbe1-injected endothelial cell accumulation at the horizontal myoseptum, as well embryos quantified and normalized over three independent as dramatic ectopic sprouting of the ISVs (Fig. 5A), as also observed experiments (Fig. 4D). We were not able to precipitate Vegfr3 using in the Tg(hsp70l:Gal4;UAS:vegfc) line (Fig. 3Cii). Interestingly, at another cross-reacting antibody directed against VEGFR3 48 hpf in double ccbe1/vegfc-overexpressing animals, we observe a intracellular domains. However, we were able to validate the dramatic accumulation of endothelial cells in dorsal aspects of the specificity of the bands and result observed by blotting lysates embryo (Fig. 5A), combined with ectopic ISV sprouting. The immunoprecipitated by the phospho-Vegfr3 antibody with the accumulation of endothelial cells (arrow) occurs at the approximate VEGFR3 antibody directed against Vegfr3 intracellular domains. We level of the FP in double Tg(shh:ccbe1;shh:vegfc), but not in single detected the bands of the same molecular weight and intensities Tg(shh:vegfc) or Tg(shh:ccbe1) animals. This could be taken to (supplementary material Fig. S5). Importantly, Ccbe1, Vegfc and indicate that Ccbe1 expression is capable of locally enhancing Vegfr3 do not cross-regulate each other transcriptionally (Fig. 4E), Vegfc-driven sprouting in the embryo. indicating that Vegfr3 expression is unchanged in this assay. This is We also examined the activity of the Tg(shh:vegfc) transgenic line consistent with immunofluorescence-based observations in the in ccbe1hu3613 mutants. ccbe1hu3613 mutant embryos showed a Ccbe1 knockout mouse (Hägerling et al., 2013). We also detected suppression of the Vegfc-driven hyperbranching of ISVs, which was normal expression of all other signaling Vegfr family members in consistent with our findings that ccbe1 is needed for Vegfc-driven the morphants examined (Fig. 4E). hyperbranching in the hsp70l:GAL4;UAS:vegfc line and dll4 depletion models. When these animals were scored at 3.5 dpf for Ccbe1 enhances Vegfc-driven sprouting endothelial cells at the horizontal myoseptum or 5 dpf for the To characterize further the function of Ccbe1 in Vegfc/Vegfr3 presence of the TD, we found that overexpression of full-length signaling, we generated two transgenic lines that express ccbe1 or Vegfc could partially rescue ccbe1hu3613 mutant lymphatic vegfc from the shh promoter in the floor plate (FP) from as early as phenotypes (supplementary material Fig. S6A,B). However, the shh- 24 hpf (supplementary material Fig. S3C,D). At 32 hpf, vegfc- or driven vegfc expression did not rescue the MO-ccbe1 lymphatic ccbe1-overexpressing animals display no phenotype. However, phenotype and, furthermore, we did not see any rescue of lymphatic when these two lines were crossed, we found that double transgenic development in MO-ccbe1-injected, hsp70l:GAL4;UAS:vegfc animals display aberrant ectopic turning of ISVs at 32 hpf (Fig. 5A). embryos (supplementary material Fig. S6C-E). We take this to

Fig. 5. Ccbe1 enhances Vegfc-driven sprouting and regulates levels of bioactive VEGFC in vitro. (A) Confocal projections at 32 hpf of Tg(shh:ccbe1), Tg(shh:vegfc) and Tg(shh:ccbe1;shh:vegfc) in a Tg(fli1a:EGFP) background. Co- overexpression of ccbe1 and vegfc in the floorplate leads to aberrant ectopic turning of the ISVs at 32 hpf (upper panels; n=32/36; P<0.0001). At 48 hpf, ccbe1 overexpression in the floorplate does not result in any phenotype, whereas vegfc-overexpressing animals display hyperbranching of the ISVs, and enhanced endothelial cell accumulation at the horizontal myoseptum (arrowhead). ccbe1 and vegfc co-overexpression in the floorplate also leads to hyperbranching ISVs, and to a marked accumulation of endothelial cells at dorsal aspects of the embryo (arrow). (B) Western blot of 293EBNA-1 cells (stably expressing VEGFC) indicate that CCBE1 is detected in the lysate of cells transfected with CCBE1 plasmid, but not in controls. (C) An increase in the levels of all forms of VEGFC is detected in the medium of CCBE1- transfected cells, compared with control cells. The mature form of VEGFC (detected at ~23 kDa) is predominant. (C′) Relative intensity (split axis) of the different processed forms of VEGFC presented in C based on multiple exposures. Note the saturation of the mature form in C. (D) qPCR showing that CCBE1 transfection does not affect VEGFC mRNA levels in vitro in 293EBNA-1 cells stably expressing VEGF-C (D, left panel). Consistent with this, in zebrafish embryos the injection of vegfc or ccbe1 mRNA does not affect the endogenous levels of the other (D, right panel). Error bars represent s.e.m. Development

1245 RESEARCH ARTICLE Development (2014) doi:10.1242/dev.100495 indicate that partial rescue exclusively in the ccbe1hu3613 mutant systems CCBE1 regulates VEGFC at a post-transcriptional level, that occurs as a result of some retained capability of the ccbe1hu3613 this regulation occurs extracellularly and that it is capable of D162E (hypomorphic) mutant protein. increasing the release and processing of VEGFC. The degree to which each of these two mechanisms apply may be dependent on the cell CCBE1 overexpression leads to enhanced levels of type, given that the effect on VEGFC release was not observed in the processed, soluble VEGFC in vitro HEK293T model system. VEGFC is produced as a secreted precursor protein requiring proteolytic cleavage of N- and C-terminal propeptides to produce the Constitutively secreted, processed VEGFC rescues ccbe1 highly bioactive mature form, consisting of dimers of the VEGF loss-of-function phenotypes homology domain (VHD), which potently activates VEGFR3 To build on these in vitro findings, and given the observation that full- signaling (Joukov et al., 1997). Although proteases that can promote length Vegfc overexpression cannot rescue the MO-ccbe1 loss-of- processing of VEGFC have been identified (McColl et al., 2003; function phenotype (supplementary material Fig. S5), we decided to Siegfried et al., 2003), other factors that might regulate the function of investigate whether a constitutively secreted, mature form of VEGFC VEGFC in the developing embryo remain to be described. To was sufficient to recue ccbe1 loss of function. To do this, we determine if the capability of CCBE1 to modulate VEGFC activity in generated a truncated form of human VEGFC (ΔNΔCVEGFC), vivo may be at the level of VEGFC production, release from the cell missing the N- and C-terminal domains but retaining the signal surface/ECM or proteolytic processing, we turned to in vitro cell-based peptide for secretion, and placed it under the control of the hsp70l models. 293EBNA-1 cells stably harboring an expression construct promoter. We used the human form because the proteolytic processing encoding full-length human VEGFC allow analysis of secreted sites of zebrafish Vegfc are not experimentally validated whereas the VEGFC polypeptides in conditioned cell culture medium. These cells human form has been examined in detail and processing sites were transfected with an expression vector encoding human CCBE1 are known (Joukov et al., 1997). We injected DNA for (Fig. 5B). In control (empty vector) or mock transfected cells lacking hsp70l:ΔNΔCVEGFC.t2a.mCherry and generated mosaic embryos. detectable CCBE1, mature VEGFC was readily detected in After a 1-hour heatshock at 26 hpf, embryos injected with conditioned cell culture media, with partially processed forms detected hsp70l:ΔNΔCVEGFC.t2a.mCherry displayed ISV hyperbranching (at at lower levels (Fig. 5C,C′). CCBE1 transfection resulted in a 54 hpf, Fig. 6A) as observed previously upon vegfc overexpression significant enrichment of all forms of VEGFC in the conditioned cell (Fig. 3C; Fig. 5A) and expressed mosaic mCherry. Co-injection of the culture media but in particular the relative abundance of the mature hsp70l:ΔNΔCVEGFC.t2a.mCherry with MO-ccbe1 generated a form was increased compared with other forms of VEGFC robust, quantifiable rescue of PL formation in these embryos (Fig. 5C,C′). This indicates that although release of VEGFC from the compared with MO-ccbe1 controls (Fig. 6A,B). Co-injecting cell surface or ECM is enhanced, processing to the mature form is hsp70l:ΔNΔCVEGFC.t2a.mCherry with MO-vegfr3 did not rescue PL particularly increased in the presence of CCBE1. formation at the horizontal myoseptum by 54 hpf (Fig. 6A,B). Finally, We confirmed the enhanced processing of VEGFC in an we found that ISV hyperbranching was present in ccbe1 and vegfr3 independent cell line, HEK293T cells, using transient transfections of morphants injected with hsp70l:ΔNΔCVEGFC.t2a.mCherry CCBE1 and VEGFC plasmids; note the increased abundance of (Fig. 6C), which is consistent with the ability of processed VEGFC to mature VEGFC in the cell culture media in response to CCBE1 in activate VEGFR2 (KDR – Human Gene Nomenclature Database) supplementary material Fig. S7B,C. However, the release of VEGFC signaling. The ability of a processed form of VEGFC to rescue the was not generally enhanced in this system as the abundance of other MO-ccbe1 loss-of-function phenotype demonstrates that a deficit in forms of VEGFC did not increase. In both cell systems, we also Vegfc maturation is responsible for venous angiogenesis and examined the levels of VEGFC present in the cell lysates and found lymphangiogenesis defects in the absence of Ccbe1. that in 293EBNA-1 cells we could detect partially processed VEGFC (i.e. the form containing the N-terminal propeptide and VEGF DISCUSSION homology domain) (supplementary material Fig. S7A) whereas in Our results show that ccbe1 genetically interacts with vegfc and HEK293T cells we detected both full-length and partially processed vegfr3 and that ccbe1 mutation or depletion suppresses the forms (supplementary material Fig. S7C). The relative levels of formation of excess and abnormal aISV and vISV sprouts driven by different forms of VEGFC in cell lysates were not markedly altered ectopic Vegfc/Vegfr3 signaling. These data highlight the necessity in the presence of CCBE1 (supplementary material Fig. S7A,C). We for functional Ccbe1 for the propagation of (ectopic) Vegfc-driven take this to indicate that the cell-associated VEGFC may be far more signals in the developing embryo. Furthermore, we show that in abundant than VEGFC in the medium, hence a small decrease in the ccbe1 and vegfr3 morphants, venous Erk signaling and Vegfr3 proportion of cell-associated VEGFC, due to release from the cell activation at Tyr1071/1076 are reduced. Our finding of a block in surface or ECM, could lead to a significant increase in the relative Vegfc/Vegfr3 signaling is consistent with phenotypes observed in abundance of VEGFC detected in the medium on western blots. humans (Alders et al., 2009) and mice (Bos et al., 2011). Previous Importantly, CCBE1 overexpression had no effect on VEGFC mRNA studies have used proximity ligation assays as a readout for Vegfr3 levels in vitro or in vivo in zebrafish embryos (Fig. 5D). signaling (Bos et al., 2011), and did not observe the reduction of In the in vitro assays above, VEGFC and CCBE1 were produced Vegfr3 signaling in the absence of Ccbe1 that we see here. However, and secreted from the same cells. To test if CCBE1 needs to be the previous study was unable to examine the entirety of signaling expressed in the same cell as VEGFC to promote VEGFC processing, in cells at equivalent developmental stages during the induction of we transfected two different populations of HEK293T cells with sprouting of lymphatics from the vein. We overcame previous CCBE1 and/or VEGFC and mixed them before detection of VEGFC limitations by utilizing the immunofluorescence visualization of in the culture medium. We found that CCBE1 was indeed capable of activated (phosphorylated) Erk in the PCV. We found that during the enhancing VEGFC processing in trans (supplementary material Fig. induction of secondary sprouting Erk activation is dorsally enriched S7B), indicating that CCBE1 can exert its effects on VEGFC outside and segmentally patterned in the PCV in a Vegfr3-dependent

the cell. These data, taken together, indicate that in these cell-based manner. This assay hence provides improved sensitivity to detect Development

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Fig. 6. Ectopic expression of mature VEGFC rescues secondary sprouting in ccbe1 morphants. (A) Confocal projections of Tg(fli1a:EGFP) at 54 hpf. Knock down of ccbe1 or vegfr3 leads to a loss of PLs at the horizontal myoseptum (arrowheads and asterisks). Ectopic expression of the mature form of VEGFC strongly rescues PL formation in ccbe1 morphants but not in vegfr3 morphants. Arrows indicate hyperbranched ISVs. (B) Quantification of PL formation at 54 hpf. In wild type, 98% (n=54/55) of embryos develop PLs, whereas in MO- ccbe1-injected embryos <4% (n=2/52) do. PL development is rescued to 74% (n=29/39) in ccbe1 morphants transiently overexpressing ΔNΔCVEGFC (P<0.0001). This rescue was never observed in vegfr3 morphants with all embryos devoid of PLs (n=23/23). (C) Quantification of ISV hypersprouting at 54 hpf. ISV hypersprouting was observed in wild-type embryos (93%; n=40/43), with mild reductions in ccbe1 morphants (79%; n=31/39) and vegfr3 morphants (65%; n=15/23) after ΔNΔCVEGFC overexpression.

Vegfc-Vegfr3 induced signaling during secondary angiogenesis in and predicts the migration route of lymphatic precursors in the trunk vivo compared with previous approaches. Using this approach and (Hogan et al., 2009a) suggesting that Ccbe1 serves to impart the also cross-reacting antibodies to phospho-Vegfr3, we found a crucial necessary post-translational activation of Vegfc (see proposed model requirement for Ccbe1 for in vivo Vegfc-Vegfr3-Erk signaling. in Fig. 7) in a spatially and temporally regulated manner. Hence, a Given this function of Ccbe1, its non-autonomous role in zebrafish migrating lymphatic endothelial cell could be directed through the (Hogan et al., 2009a) and its extracellular localization (Bos et al., embryonic environment by a route pre-determined by the earlier, 2011), we asked if Ccbe1 can directly modulate the levels of bioactive local concentration of Ccbe1. To test this idea directly is inherently Vegfc. We did this in cell-based models, and found that upon CCBE1 difficult, but the hypothesis is supported by overexpression regimes overexpression, the amount of mature bioactive VEGFC is increased of ccbe1 and vegfc in an ectopic location, the FP of the neural tube: in the culture medium. CCBE1 can induce higher levels of VEGFC here we found that Vegfc-driven sprouting of endothelial cells was in trans and hence performs this function outside of the cell in this in enhanced in the presence of ectopic Ccbe1. vitro context. Fully processed VEGFC was increased more than the partially processed and full-length forms in the medium, indicating that CCBE1 regulates processing. However, CCBE1 can increase levels of the partially processed and full-length forms to some degree, also suggesting enhanced release from the cell surface or matrix. Confirming that this capability has relevance in vivo, we observed a rescue of the ccbe1 loss-of-function phenotype when we reintroduced a secreted and fully processed form of VEGFC in zebrafish. This rescue, combined with the observation that full-length Vegfc failed to rescue the phenotype, demonstrates that Ccbe1 regulates Vegfc maturation and bioavailability. Ccbe1 does not appear to be a protease and the precise molecular mechanisms involved, including the roles of additional proteins in the CCBE1/VEGFC/VEGFR3 pathway, require further analysis. One pertinent question is: why would the developing embryo need an additional factor to regulate Vegfc-driven activation of Vegfr3? It is crucial to note that vegfc in zebrafish is transcribed in Fig. 7. Ccbe1 activates Vegfc to induce Vegfr3 signaling. Proposed the hypochord and dorsal aorta in the midline of the embryonic model for coordination of angiogenesis by Ccbe1, Vegfc and Vegfr3 in the trunk. Despite this, secondary sprouts from the vein migrate past this developing embryo. Vegfc is produced in a largely inactive full-length form transcriptional source of ligand on the dorsoventral and mediolateral that is processed and released from the cell surface/ECM in a Ccbe1- axes of the embryo to the horizontal myoseptum and give rise to dependent manner to generate the mature, highly active form. Downstream, arteries respond in a manner dampened by Dll4-dependent suppression of PLs. This suggests that spatial presentation of active Vegfc protein Vegfr3 signaling (Hogan et al., 2009b), whereas Vegfr3 signaling in veins during secondary sprouting must be regulated independently of the induces secondary angiogenesis, which produces both intersegmental veins

transcriptional source of vegfc. ccbe1 transcript expression precedes and lymphatic vascular precursor cells. Development

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CCBE1 mutations have been shown to be responsible for with both anti-phospho-VEGFR3 (1/1000; Cell Applications, CB5793) and generalized lymph vessel dysplasia in humans (Alders et al., 2009; anti (non-phosphorylated) VEGFR3 (1/1000; Cell Applications, CB5792). Connell et al., 2010). Our finding that Ccbe1 is a crucial modulator Mouse anti-myosin light chain 1 and 3f (1/100; Developmental Studies of the Vegfc/Vegfr3 pathway during embryo development, suggests Hybridoma Bank, F310) was used to detect a loading control band. that a deficiency of VEGFR3 signaling may be responsible for the Quantification relative to IgG light chain (phospho-Vegfr3 only) or Myosin used ImageJ across three biological replicates. Only phospho-Vegfr3 was lymphatic aspects of Hennekam Syndrome. Other aspects of this assessed and not total Vegfr3, owing to a limitation of the cross-reacting syndrome that are distinct from Milroy’s disease might be due to antibodies in only detecting bands post-IP for phospho-Vegfr3. other, yet to be identified, functions of CCBE1. VEGFC processing/secretion in vitro MATERIALS AND METHODS For analysis of VEGFC processing and secretion two approaches were used. Zebrafish strains and transgenesis Briefly, using 293EBNA-1 cells stably expressing a full-length form of Animal work followed guidelines of the animal ethics committees at the human VEGFC (Karnezis et al., 2012) were transiently transfected University of Queensland, and the Royal Netherlands Academy of Arts and [Lipofectamine 2000 (Invitrogen)] with a CCBE1 expression vector after a Sciences (DEC). Zebrafish transgenic lines Tg(fli1a:EGFP), Tg(−6.5kdr- medium change to a serum-free chemically defined medium (Pro293a- l:mCherry), Tg(−0.8flt1:tdTomato) and Tg(hsp70l:Gal4)1.5kca4 lines were CDM, Lonza). Both supernatant and cell lysates were subsequently collected described previously (Scheer et al., 2001; Lawson and Weinstein, 2002; for western blotting. 293EBNA-1 cells were maintained in Dulbecco’s Hogan et al., 2009a; Bussmann et al., 2010). N-ethyl-N-nitrosourea (ENU) Modified Eagle Medium, supplemented with 10% fetal bovine serum, mutagenesis was performed as previously described (Wienholds et al., 2002). 50 mM L-glutamine, 50 μg/ml penicillin, 50 μg/ml streptomycin and The Tg(4xUAS:vegfc) line was generated using a 4xUAS promoter 100 μg/ml hygromycin B (Roche Diagnostics). Cells were lysed in 0.1% upstream of the full length zebrafish vegfc cDNA (supplementary material SDS, 50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, Fig. S3A) cloned using the Gateway system (Hartley et al., 2000; Walhout pH 8.0, 1 mM PMSF, 10 μg/ml aprotinin and 10 μg/ml leupeptin. Lysates et al., 2000). The Tg(flt4:YFP) reporter line was generated from BAC were incubated at 4°C for 20 minutes with gentle agitation and centrifuged DKEY-58G10 as previously described (Bussmann and Schulte-Merker, at 13,000 g for 10 minutes. Western blotting was performed with antibodies 2011) and is characterized in detail elsewhere (van Impel et al., 2014). The targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967). Tg(s1173:Gal4) line was kindly provided by Ethan Scott (School of For cell mixing experiments, separate populations of HEK293T cells were Biomedical Sciences, University of Queensland). sonic hedgehog (shh) transfected with constructs expressing CCBE1, VEGFC or both. Cells were promoter and floorplate enhancer (Ertzer et al., 2007) was used to drive passaged and mixed as indicated after 24 hours. From mixed cultures, Ccbe1 IRES tagRFP or Vegfc IRES mturquoise (supplementary material conditioned supernatants were collected and cells were lysed in RIPA/SDS Fig. S3C), cloned into the MiniTol2 vector (Balciunas et al., 2006); plasmids buffer after another 48 hours. Supernatant and cell lysates were isolated and (25 ng/μl) were injected with tol2 transposase mRNA (25 ng/μl) into 1- to western blotting analysis performed using VEGFC (VEGFC isoform 103 2-cell-stage embryos. All genotyping details and primers are given in antibody, antibodies-online) and CCBE1 (HPA041374, Sigma) antibodies. supplementary material Tables S1 and S3. KASPar (KBioscience) was used for the indicated vegfc alleles. qPCR detection of Vegfc and Ccbe1 expression For expression analysis in the cultured cells, RNA was isolated using DNA, RNA and morpholino injections RNeasy Mini Kit (Qiagen), and cDNA synthesized using High Capacity Constructs for transient DNA injection of the cDNA Reverse Transcription Kit (Applied Biosystems). For expression Tg(hsp70l:ΔΔVEGFC.t2a.mCherry) transgene were generated by PCR analysis, Taqman Gene Expression Assays were employed [VEGFC amplifying the N- and C-terminally truncated form of human VEGFC, (Hs01099203_m1), GAPDH (Mm99999915_g1)] using Taqman Fast containing the endogenous secretion peptide (Joukov et al., 1997) followed by Universal PCR Master Mix (2×) (Applied Biosystems) as per Gateway recombineering. Primer sequences are given in supplementary manufacturer’s instructions and analyzed on a 7300 RT-PCR machine material Table S2. DNA was injected at a concentration of 90 ng/μl at single- (Applied Biosystems). For zebrafish qPCR, RNA was extracted at 24 hpf cell stage. Morpholinos used are shown in supplementary material Table S4. from 20 zebrafish embryos. cDNA was synthesized using the Superscript III The vegfc cDNA used was described previously (Hogan et al., 2009a). Kit (Invitrogen) and reactions used SYBR Green kit (Applied Biosystems) analyzed on a 7500 RT-PCR machine (Applied Biosystems). Data were Analysis of ERK and Vegfr3 signaling in zebrafish embryos normalized using the geometric average of ef1α (eef1a1l1), rpl13 and rps29, Phospho-ERK was detected by immunofluorescence, using the previously which were found to be the most stable reference genes using GeNorm with described protocol (Inoue and Wittbrodt, 2011) but modified as follows: data displayed as arbitrary units (A.U.). embryos were blocked and incubated with p-ERK primary antibody (1/250; Cell Signaling, #4370) in TBS containing 0.1% Triton X-100, 1% bovine Imaging serum albumin, 10% goat serum and then with horseradish peroxidase- Confocal imaging was performed on live embryos mounted laterally using conjugated anti-goat secondary antibody (1/1000; Cell Signaling, #7074) in a Zeiss 510 or a Leica SPE confocal microscope at the indicated stages. 2% blocking solution (Roche) dissolved in 100 mM maleic acid with 150 Sections were imaged using a Zeiss 710 FCS confocal microscope. mM NaCl (pH 7.4). Phospho-ERK was subsequently detected using the TSA reagent (Perkin Elmer) as per manufacturer’s guidelines. As a control Statistical analysis and mutation effect prediction for antibody specificity, embryos were incubated in 100 μM PD98059 (Cell For genetic interaction data, χ2 tests were performed using GraphPad Signaling) from 24 hpf. Embryos were vibratome sectioned (100 μm) (Leica (http://graphpad.com/quickcalcs/chisquared1.cfm). PolyPhen-2 was used to VT100S) and imaged as described below. evaluate mutation pathogenicity (http://genetics.bwh.harvard.edu/pph2/). Zebrafish phospho-Vegfr3 was detected following immunoprecipitation (IP) with anti phospho-VEGFR3 (Cell Applications, CB5793) using the Acknowledgements same antibody. One hundred and fifty embryos were lysed overnight at 4°C We thank C. Neyt, G. van de Hoek, N. Chrispijn and M. Witte for technical in modified RIPA buffer containing 1 mM EDTA, 4% protease inhibitor assistance; Dagmar Wilhelm for advice; Nathan Lawson for providing an initial P- (Sigma), 1% phenylmethylsulfonyl fluoride (PMSF; Sigma) and 1% Halt Erk immunofluorescence protocol; and Ethan Scott for providing the Tg(s1173:Gal4) line. Imaging was performed in the Australian Cancer Research Foundation’s phosphatase inhibitor cocktail (Thermo Fisher Scientific). IP was carried out Dynamic Imaging Facility at IMB and at the Hubrecht Imaging Center (HIC). overnight at 4°C with the phospho-Vegfr3 antibody (1/1000), followed by 2 hours at 4°C with protein A agarose beads (1/10) (Thermo Fisher Competing interests

Scientific). Standard western blotting approaches were used to detect Vegfr3 The authors declare no competing financial interests. Development

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Author contributions Irrthum, A., Devriendt, K., Chitayat, D., Matthijs, G., Glade, C., Steijlen, P. M., L.L.G. designed and performed experiments, analyzed data and co-wrote the Fryns, J. P., Van Steensel, M. A. and Vikkula, M. (2003). Mutations in the manuscript; T.K., D.S., N.C.H., K.K., G.R., N.I.B. and A.v.I. designed and transcription factor gene SOX18 underlie recessive and dominant forms of performed experiments, analyzed data and edited the manuscript; S.A.S. and hypotrichosis-lymphedema-telangiectasia. Am. J. Hum. Genet. 72, 1470-1478. Isogai, S., Lawson, N. D., Torrealday, S., Horiguchi, M. and Weinstein, B. M. M.G.A. designed experiments, analyzed data and edited the manuscript; S.S.-M. (2003). Angiogenic network formation in the developing vertebrate trunk. and B.M.H. designed experiments, analyzed data and co-wrote the manuscript. Development 130, 5281-5290. Isogai, S., Hitomi, J., Yaniv, K. and Weinstein, B. M. (2009). Zebrafish as a new Funding animal model to study lymphangiogenesis. Anat. Sci. Int. 84, 102-111. This work was supported by an Australian Research Council Future Fellowship Jeltsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., Rauvala, H., Swartz, [FT100100165 to B.M.H.]; a European Molecular Biology Organization Long-Term M., Fukumura, D., Jain, R. K. and Alitalo, K. (1997). Hyperplasia of lymphatic Fellowship [LTRF 52-2007 to T.K.]; Veni grants from The Netherlands Organisation vessels in VEGF-C transgenic mice. Science 276, 1423-1425. for Scientific Research (NWO) [916.10.132 to T.K.; 863.11.022 to G.R.]; Marie Joukov, V., Sorsa, T., Kumar, V., Jeltsch, M., Claesson-Welsh, L., Cao, Y., Saksela, O., Kalkkinen, N. and Alitalo, K. (1997). Proteolytic processing regulates receptor Curie Intra-European Fellowships (to D.S. and A.v.I.); National Health and Medical specificity and activity of VEGF-C. EMBO J. 16, 3898-3911. Research Council of Australia (NHMRC) project grants [631657 and Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V. W., Fang, G. H., APP1050138]; a Program Grant [487900 to M.G.A. and S.A.S.] and Research Dumont, D., Breitman, M. and Alitalo, K. (1995). Expression of the fms-like Fellowships (to M.G.A. and S.A.S.) from the National Health and Medical tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during Research Council; and KNAW (S.S.-M.). development. Proc. Natl. Acad. Sci. USA 92, 3566-3570. Karkkainen, M. J., Ferrell, R. E., Lawrence, E. C., Kimak, M. A., Levinson, K. L., Supplementary material McTigue, M. A., Alitalo, K. and Finegold, D. N. (2000). Missense mutations Supplementary material available online at interfere with VEGFR-3 signalling in primary lymphoedema. Nat. Genet. 25, 153-159. Karkkainen, M. J., Haiko, P., Sainio, K., Partanen, J., Taipale, J., Petrova, T. 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