Download Ccbe1 regulates Vegfc-mediated induction of Vegfr3

© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1239-1249 doi:10.1242/dev.100495
RESEARCH ARTICLE
Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling
during embryonic lymphangiogenesis
ABSTRACT
The VEGFC/VEGFR3 signaling pathway is essential for
lymphangiogenesis (the formation of lymphatic vessels from preexisting vasculature) during embryonic development, tissue
regeneration and tumor progression. The recently identified secreted
protein CCBE1 is indispensible for lymphangiogenesis during
development. The role of CCBE1 orthologs is highly conserved in
zebrafish, mice and humans with mutations in CCBE1 causing
generalized lymphatic dysplasia and lymphedema (Hennekam
syndrome). To date, the mechanism by which CCBE1 acts remains
unknown. Here, we find that ccbe1 genetically interacts with both
vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by
increased Vegfc are suppressed in the absence of Ccbe1, and Vegfcdriven sprouting is enhanced by local Ccbe1 overexpression.
Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in
the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the
levels of fully processed, mature VEGFC in vitro and the
overexpression of mature VEGFC rescues ccbe1 loss-of-function
phenotypes in zebrafish. Taken together, these data identify Ccbe1
as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.
KEY WORDS: Angiogenesis, Lymphangiogenesis, Lymphedema,
Vasculature, Zebrafish
INTRODUCTION
The lymphatic vasculature develops from pre-existing vessels in a
dynamic process called lymphangiogenesis, and is necessary to
preserve tissue fluid homeostasis, for fat absorption and normal
immune function. The lymphatic vascular network originates in the
mouse embryo from the cardinal vein, where lymphatic endothelial
precursor cells first bud and migrate dorsolaterally away from the vein
under the control of Vegfc and its receptor Vegfr3 (Flt4 – Mouse
Genome Informatics). In mice, the overexpression of Vegfc in the skin
results in dramatic hyperplasia of lymphatic vessels (Jeltsch et al.,
1997), whereas Vegfc knockout mice develop lymphatic hypoplasia
and lymphedema (Karkkainen et al., 2004). In developing Vegfc
knockout embryos, endothelial cells are still specified to the lymphatic
1
Division of Molecular Genetics and Development, Institute for Molecular
Bioscience, The University of Queensland, St Lucia, QLD 4073, Australia.
2
Hubrecht Institute – KNAW & UMC Utrecht, 3584 CT Utrecht, The Netherlands.
3
Tumour Angiogenesis Programme, Peter MacCallum Cancer Centre, Locked Bag
1, A’Beckett Street, Melbourne, VIC 8006, Australia. 4Sir Peter MacCallum
Department of Oncology, University of Melbourne, Parkville, VIC 3010, Australia.
5
EZO, WUR, 6708LX Wageningen, The Netherlands.
*Present address: U1046, Université Montpellier 1, Université Montpellier 2, 34967
CEDEX 2 Montpellier, France.
‡
These authors contributed equally to this work
§
Author for correspondence ([email protected])
Received 26 June 2013; Accepted 30 December 2013
lineage, and express Prox1, but fail to migrate from the cardinal vein
(Karkkainen et al., 2004; Hägerling et al., 2013). Reduced
development, or impaired function of lymphatics, leads to tissue fluid
accumulation and lymphedema in humans, which can be inherited as
a result of mutations in key developmental genes (Karkkainen et al.,
2000; Irrthum et al., 2003; Alders et al., 2009; Connell et al., 2010).
A mutation in VEGFC has recently been shown to be responsible for
inherited lymphedema (Gordon et al., 2013).
Vegfr3-deficient mice die early during gestation (embryonic day
9.5) owing to cardiovascular failure (Dumont et al., 1998),
consistent with the expression of Vegfr3 in blood vascular
endothelium in the early embryo. However, after midgestation,
Vegfr3 expression becomes enriched in the developing lymphatic
vasculature (Kaipainen et al., 1995) and Vegfr3 signaling is
sufficient to initiate lymphangiogenesis (Veikkola et al., 2001).
Importantly, patients with mutations in VEGFR3 (FLT4 – Human
Gene Nomenclature Database) develop Milroy’s disease,
characterized by reduced lymphatic drainage and lymphedema in the
lower limbs (Karkkainen et al., 2000). These studies, and many
more, have shown that the precise modulation of the Vegfc/Vegfr3
signaling pathway is crucial during lymphatic vascular development.
In zebrafish, lymphatic vascular development initiates from
32 hours post-fertilization (hpf), when precursor cells first migrate
dorsally from the cardinal vein to colonize the horizontal
myoseptum, generating a population of parachordal
lymphangioblasts (PLs) (Küchler et al., 2006; Yaniv et al., 2006;
Hogan et al., 2009a; Isogai et al., 2009). These PLs subsequently
migrate alongside arteries, both dorsally and ventrally (from ~60
hpf), and remodel into the major trunk lymphatic vessels (Bussmann
et al., 2010; Cha et al., 2012), forming the thoracic duct,
intersegmental lymphatic vessels and dorsal longitudinal lymphatic
vessels (for reviews, see Koltowska et al., 2013; van Impel and
Schulte-Merker, 2014). In addition, a complex craniofacial
lymphatic network is formed (Okuda et al., 2012). Zebrafish vegfr3
(flt4 – Zebrafish Information Network) and vegfc function is
essential for all secondary (venous) angiogenesis, including the
sprouting of lymphatic precursors from the cardinal vein and
subsequent formation of PLs (Isogai et al., 2003; Küchler et al.,
2006; Yaniv et al., 2006; Hogan et al., 2009a).
We have previously reported two zebrafish mutants with an
absence of lymphatic (but not blood vascular) development (Hogan
et al., 2009a; Hogan et al., 2009b). These phenotypes were caused
by mutations in vegfr3 and the previously uncharacterized gene
collagen and calcium-binding EGF domains-1 (ccbe1) (Hogan et
al., 2009a; Hogan et al., 2009b). ccbe1 was shown to act at stages
identical to vegfr3 and vegfc (Hogan et al., 2009a). ccbe1 encodes
an extracellular matrix (ECM) protein, and is composed of Nterminal calcium-binding epidermal growth factor (EGF)-like and
EGF domains, and two collagen-repeat domains towards the C
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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,‡,§
Fig. 1. See next page for legend.
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RESEARCH ARTICLE
Fig. 1. Zebrafish vegfc mutants lack lymphatic vessels, but appear
otherwise normal. (A-D) Confocal projections of Tg(fli1a:EGFP) show
unaltered overall morphology and blood vasculature in vegfchu5055 mutants
(B) compared with wild type (A). The TD (C, arrows) is absent in vegfchu5055
(D, asterisks). (E) Positional cloning linked alleles hu6124 and hu5055 to
large regions of chromosome 1, containing the vegfc gene. For hu6124, 13
recombinant embryos identified at z9394 were reduced to four recombinant
embryos common with z5508, identifying linkage in a large region containing
vegfc. For hu5055, eight recombinant embryos at z2691 and 33 different
recombinant embryos at z4694 flanked a large region of chromosome 1, also
containing vegfc. (F) Sequencing of vegfc alleles. hu6410 encodes an early
stop allele leading to a predicted truncated protein lacking the core (VHD)
region. hu5142, hu6124 and hu5055 encode missense mutations, all in
highly conserved regions of the protein (G) and predicted to be damaging by
PolyPhen-2 (Adzhubei et al., 2010) with scores of 1 out of 1. (G) Alleles
hu5055, hu6410, hu6124 and hu5142 all encode mutations in regions that
are conserved across multiple species. (H) Alleles hu5055 and hu6410 fail to
complement, with trans-heterozygote embryos displaying a lack of lymphatic
structures; phenotype scored as percentage extent of thoracic duct over ten
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)
or TD extent (J). The hu6410 allele (L107X) shows the most severe defects
in heterozygous and homozygous mutants, whereas hu5055 does not show
haplo-insufficiency phenotypes. (K) vegfchu5055 genetically interacts with
vegfr3hu4602 in trans-heterozygous embryos. Confocal projections of
Tg(fli1a:EGFP; kdr-l:mCherry) showing examples of the heterogeneity
observed during TD development at 5 dpf in vegfc+/−; vegfr3+/− embryos.
Arrows indicate the thoracic duct, asterisks indicate absence of thoracic duct.
terminus of the protein. The protein localizes to secretory vesicles
(Alders et al., 2009), and is secreted to the ECM, where it binds
proteins such as collagens or vitronectin (Bos et al., 2011).
Consistently, Ccbe1 functions non-cell-autonomously in zebrafish
(Hogan et al., 2009a). There are no described receptors or pathways
known to interact with Ccbe1, with progress limited at least in part
by the fact that full-length CCBE1 protein is insoluble in many
standard assays (S.S.-M. and B.M.H., unpublished observations).
Importantly, mice deficient for Ccbe1 lack lymphatic vasculature
(Bos et al., 2011) and mutations in human CCBE1 are causative for
generalized lymphedema and lymphangiectasia in Hennekam
syndrome (Alders et al., 2009; Connell et al., 2010). Interestingly,
treatment with Ccbe1 protein (a truncated soluble form) increased
Vegfc-induced lymphangiogenesis in a cornea micropocket assay (Bos
et al., 2011). Furthermore, mice heterozygous for both Vegfc and
Ccbe1 show a genetic interaction in lymphatic development
(Hägerling et al., 2013). These results were suggestive that Ccbe1 and
Vegfc can function together in lymphangiogenesis, but mechanistic
insights into the molecular function of Ccbe1 have remained elusive.
Here, we have investigated the relationship between Ccbe1 and the
Vegfc/Vegfr3 signaling pathways. Using genetic epistasis and analysis
of signaling events in vivo, we show that the embryo requires Ccbe1
for normal Vegfc/Vegfr3/Erk signaling. CCBE1 is capable of
increasing the levels of mature, processed VEGFC in vitro, which can
rescue ccbe1 loss-of-function phenotypes when overexpressed in vivo.
Together, our findings suggest that Ccbe1 activates Vegfc through its
processing and release from the ECM in order to regulate
lymphangiogenesis and venous sprouting in the embryo. These
findings suggest common mechanisms in development and vascular
pathologies (e.g. lymphedema) and further suggest Ccbe1 as a new
therapeutic entry point in treating pathological lymphangiogenesis.
RESULTS
Identification of four zebrafish vegfc mutant alleles
In a forward genetic screen for zebrafish lymphatic vascular
mutants, we identified several mutants that were indistinguishable
Development (2014) doi:10.1242/dev.100495
from vegfr3 and ccbe1 mutants at the level of gross embryo
morphology (supplementary material Fig. S1A-D), and that
displayed a severe reduction in secondary angiogenesis and block in
lymphatic precursor sprouting from the cardinal vein (Fig. 1A-D;
Fig. 2A-H; supplementary material Fig. S1E-K). At 5 days postfertilization (dpf), the hu5055, hu6124, hu5142 and hu6410 mutants
have a grossly normal, functional blood vasculature, but the
lymphatic vasculature is absent (Fig. 1B,D). Using a positional
cloning approach, we found that these mutants were linked to
chromosome 1, containing the vegfc gene (Fig. 1E; data not shown).
Sequencing of vegfc in hu5055 mutants revealed a missense
mutation changing a cysteine into an arginine residue (C365R) in
the highly conserved C-terminal propeptide (Fig. 1F,G). Additional
mutations identified caused a premature stop in hu6410 (L107X), a
phenylalanine to cysteine change in the highly conserved VHDVEGF homology domain (F138C) and another cysteine-to-serine
substitution in the C-terminal propeptide of Vegfc (C339S)
(Fig. 1F,G). All mutations were predicted to be damaging by
PolyPhen-2 (Adzhubei et al., 2010). These vegfc mutations
segregate with the thoracic duct (TD) deficiency phenotype and
phenocopy MO-vegfc knockdown or soluble Vegfr3 ligand trapinduced phenotypes (Küchler et al., 2006; Yaniv et al., 2006; Hogan
et al., 2009a; Hogan et al., 2009b). Furthermore, these phenotypes
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
variable penetrance of different alleles and haplo-insufficient
phenotypes for the more penetrant alleles (Fig. 1I,J). The hu5055
allele segregated in a Mendelian, autosomal recessive manner and was
thus used in subsequent assays. Crossing hu5055 mutant carriers to
the previously described expando/vegfr3 mutant (Hogan et al., 2009b),
we found an increased frequency of TD defects consistent with
genetic interaction in the same pathway (Fig. 1K; Fig. 2I).
ccbe1, vegfr3 and vegfc zebrafish mutants genetically
interact
The ccbe1hu3613, vegfr3hu4602 and vegfchu5055 mutants display a
complete loss of the lymphatic vasculature when homozygous
(Fig. 2A-H). Previous, non-quantitative observations (Hogan et al.,
2009a; Hogan et al., 2009b) indicated that partial loss of ccbe1 or
vegfr3 led to phenotypically wild-type lymphatic development. We
took advantage of these mutants to determine more rigorously if
combined heterozygous mutations led to enhanced lymphatic
phenotypes. We crossed heterozygous carriers and quantified the
extent of the TD across ten body segments in the trunks of
individual embryos. Subsequent genotyping first confirmed the
genetic interaction in vegfchu5055/+, vegfr3hu4602/+ double
heterozygous animals (Fig. 2I). We found that ~28% of scored
embryos displayed ≤50% TD extent; within this population, 71% of
the embryos were double heterozygotes for vegfc and vegfr3, a
significant enrichment from total double heterozygosity of 27%
(Fig. 2I). Importantly, similar robust interactions of vegfr3 and vegfc
were observed with ccbe1. The population of embryos displaying
≤50% TD extent from a ccbe1hu3613/+, vegfr3hu4602/+ cross was
enriched (54%) for double heterozygotes, as were embryos from a
ccbe1hu3613/+, vegfchu5055/+ cross (61%) (Fig. 2J,K). This genetic
interaction seems to be selective to the ccbe1hu3613, vegfr3hu4602 and
vegfchu5055 mutants because in crosses to two other, as yet genetically
uncharacterized lymphatic mutants, we did not observe any
interaction (L.L.G. and B.M.H., unpublished observations). Finally,
we generated triple heterozygote embryos and found that the
population developing ≤50% TD extent contained most of the triple
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heterozygote embryos, demonstrating a strong interaction (Fig. 2L).
This synergistic genetic interaction shows that lymphatic
development is sensitive to the level of activation of the Vegfc/
Vegfr3 pathway, and that altering ccbe1 dosage modifies phenotypes
in this pathway. Combined with the fact that ccbe1, vegfc and vegfr3
act at the same stage during the cellular progression of
lymphangiogenesis and that these mutants present the most selective
loss of lymphatic vascular structures that we have observed, we
considered this a strong indication that Ccbe1 could be a novel
component of the Vegfc/Vegfr3 signaling pathway.
ccbe1 mutation suppresses phenotypes driven by ectopic
Vegfc/Vegfr3 signaling in embryonic arteries
Previous studies have shown that the knockdown of dll4 leads to an
excessive intersegmental artery (aISV) angiogenesis phenotype by 72
hpf (Leslie et al., 2007; Siekmann and Lawson, 2007; Hogan et al.,
2009b). This phenotype occurs because in wild-type arteries, the
Vegfc/Vegfr3 pathway is inhibited by Dll4. Depletion of Dll4 leads to
increased activation of Vegfc/Vegfr3 signaling in aISVs, resulting in
aISV hyperbranching (Leslie et al., 2007; Siekmann and Lawson,
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2007; Hogan et al., 2009b). We took advantage of the hyperbranching
phenotype to investigate ccbe1 function. In clutches of embryos
derived from incrosses of ccbe1 heterozygous carriers, we activated
the Vegfc/Vegfr3 pathway in arteries by knocking down dll4.
Embryos were first sorted at 72 hpf according to the severity of
arterial hyperbranching, and then subsequently genotyped. The
population containing animals with vastly reduced or no
hyperbranching (Fig. 3Aiii, graph) was significantly enriched in
ccbe1hu3613 mutants (81%; P<0.0001) whereas the category containing
severe aISV hyperbranching (Fig. 3Aii) was enriched in wild-type and
heterozygote embryos (83%; P=0.0019; Fig. 3A, graph). Hence,
ccbe1 mutation suppressed the dll4 loss-of-function phenotype.
To validate this observation further, we used a second phenotypic
assay that has been previously described (Hogan et al., 2009b). When
vegfc is overexpressed by mRNA injection and dll4 is simultaneously
depleted, we observe ectopic turning of aISVs in the trunk from as
early as 30 hpf (compare Fig. 3Bi with 3Bii) that is not seen in single
treatment controls (supplementary material Fig. S2). aISVs deficient
for dll4 are more responsive to vegfc RNA injection (Hogan et al.,
2009b) and phenotypes appear the same as those observed when vegfc
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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; kdrl: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%.
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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.
Ccbe1 knockdown suppresses phenotypes driven by ectopic
Vegfc/Vegfr3 signaling in embryonic veins
We next generated a new zebrafish transgenic line
(hsp70l:GAL4;UAS:vegfc), which ubiquitously expresses full-length
vegfc under the control of the heat shock (hsp70l) promoter
(supplementary material Fig. S3A,B) (Scheer et al., 2001). In these
embryos (following two consecutive heat shocks at 28 and 56 hpf),
veins sprout ectopically, resulting in hyperbranched intersegmental
vessels (vISVs) as visualized in fli1a:GFP, flt1:tomato double
transgenic animals (Fig. 3Ciii). ccbe1 morpholino injection rescued
this phenotype, blocking excessive venous sprouting (Fig. 3Civ,
graph; P<0.0001).
Taken together, we found in two blind, genotype-based
experiments, that Vegfc- and Vegfr3-driven arterial phenotypes are
suppressed in ccbe1hu3613 mutants, and additionally that a Vegfc- and
Vegfr3-driven venous phenotype is rescued in ccbe1 morphant
embryos. These findings suggest that Ccbe1 is necessary for the
function of the Vegfc/Vegfr3 signaling pathway during embryonic
development.
Vegfr3-dependent Erk activation in embryonic veins
requires ccbe1
It is well established that VEGFR3 signals via intracellular kinases
that include ERK (reviewed by Bahram and Claesson-Welsh, 2010).
We decided to further investigate downstream signaling pathways
to determine if Ccbe1-deficient embryos display a signaling block
in venous endothelium. We used whole-mount immunofluorescence
to examine phospho-Erk in the context of the embryonic
vasculature. The signal observed was specific as it was reduced in
embryos treated with the MEK inhibitor PD98059 (which inhibits
Mek activation and hence Erk phosphorylation) and was ectopically
induced in the ventral posterior cardinal vein (PCV) in the context
of vegfc overexpression (Fig. 4A). In wild-type embryos, we found
that Erk activity was broadly detected in the neural tube, muscle and
epithelia, but also in the endothelium of the PCV. In PCV
endothelium, Erk activation was segmented and dorsally enriched
in individual cells at 32 hpf, concomitant with the induction of
secondary sprouting. Importantly, this patterned activation of Erk
was markedly reduced in both MO-vegfr3- and MO-ccbe1-injected
embryos compared with wild-type controls (Fig. 4B,C). These
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is expressed from the hsp70l promoter (Nicoli et al., 2012). In clutches
of embryos derived from ccbe1 heterozygous incrosses, we
ectopically activated the Vegfc/Vegfr3 pathway by injecting vegfc
mRNA and a dll4 morpholino. Animals were sorted at 30 hpf
according to the severity of aISV phenotype, and then genotyped. Of
the embryos displaying a weak phenotype, or no phenotype at all
(Fig. 3Biii), 70% were ccbe1 mutants (Fig. 3B, graph), a highly
significant (P<0.0001) enrichment. The population displaying severe
phenotypes was significantly enriched in wild-type and heterozygote
animals (83%; P=0.0006; Fig. 3B, graph). Hence, ccbe1 mutation
suppressed the dll4 loss-of-function, vegfc overexpression phenotype.
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Development (2014) doi:10.1242/dev.100495
findings confirm the presence of Vegfc- and Vegfr3-dependent Erk
signaling in embryonic veins and demonstrate that Ccbe1 is
necessary for activation of this pathway.
We also examined the activation status of Vegfr3 by western blot
of embryonic lysates. The phosphorylation of Tyr1063/1068 in
human VEGFR3 reflects the activation status of VEGFR3,
promoting downstream signaling essential for lymphangiogenesis
(Dixelius et al., 2003; Salameh et al., 2005). Antibodies against
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zebrafish Vegfr3 are unavailable, but we found two commercial
antibodies directed against conserved regions of human VEGFR3
that cross-reacted with zebrafish Vegfr3. One of these was directed
against the phosphorylated residues Tyr1063/1068 (human) or
Tyr1071/1076 in zebrafish (supplementary material Fig. S4). Using
this phospho-VEGFR3 antibody for western blotting we detected
Vegfr3 in zebrafish lysates immunoprecipitated with the same
antibody at 32 hpf, during initiation of secondary sprouting. The
Development
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).
signal was increased in vegfc mRNA-injected embryos, but was
reduced in MO-vegfr3- and MO-vegfc-injected embryos. A similar
reduction in phospho-Vegfr3 was observed in MO-ccbe1-injected
embryos quantified and normalized over three independent
experiments (Fig. 4D). We were not able to precipitate Vegfr3 using
another cross-reacting antibody directed against VEGFR3
intracellular domains. However, we were able to validate the
specificity of the bands and result observed by blotting lysates
immunoprecipitated by the phospho-Vegfr3 antibody with the
VEGFR3 antibody directed against Vegfr3 intracellular domains. We
detected the bands of the same molecular weight and intensities
(supplementary material Fig. S5). Importantly, Ccbe1, Vegfc and
Vegfr3 do not cross-regulate each other transcriptionally (Fig. 4E),
indicating that Vegfr3 expression is unchanged in this assay. This is
consistent with immunofluorescence-based observations in the
Ccbe1 knockout mouse (Hägerling et al., 2013). We also detected
normal expression of all other signaling Vegfr family members in
the morphants examined (Fig. 4E).
Ccbe1 enhances Vegfc-driven sprouting
To characterize further the function of Ccbe1 in Vegfc/Vegfr3
signaling, we generated two transgenic lines that express ccbe1 or
vegfc from the shh promoter in the floor plate (FP) from as early as
24 hpf (supplementary material Fig. S3C,D). At 32 hpf, vegfc- or
ccbe1-overexpressing animals display no phenotype. However,
when these two lines were crossed, we found that double transgenic
animals display aberrant ectopic turning of ISVs at 32 hpf (Fig. 5A).
Development (2014) doi:10.1242/dev.100495
At 48 hpf, ccbe1-overexpressing animals still show no phenotype,
whereas vegfc-overexpressing animals display vastly increased
endothelial cell accumulation at the horizontal myoseptum, as well
as dramatic ectopic sprouting of the ISVs (Fig. 5A), as also observed
in the Tg(hsp70l:Gal4;UAS:vegfc) line (Fig. 3Cii). Interestingly, at
48 hpf in double ccbe1/vegfc-overexpressing animals, we observe a
dramatic accumulation of endothelial cells in dorsal aspects of the
embryo (Fig. 5A), combined with ectopic ISV sprouting. The
accumulation of endothelial cells (arrow) occurs at the approximate
level of the FP in double Tg(shh:ccbe1;shh:vegfc), but not in single
Tg(shh:vegfc) or Tg(shh:ccbe1) animals. This could be taken to
indicate that Ccbe1 expression is capable of locally enhancing
Vegfc-driven sprouting in the embryo.
We also examined the activity of the Tg(shh:vegfc) transgenic line
in ccbe1hu3613 mutants. ccbe1hu3613 mutant embryos showed a
suppression of the Vegfc-driven hyperbranching of ISVs, which was
consistent with our findings that ccbe1 is needed for Vegfc-driven
hyperbranching in the hsp70l:GAL4;UAS:vegfc line and dll4
depletion models. When these animals were scored at 3.5 dpf for
endothelial cells at the horizontal myoseptum or 5 dpf for the
presence of the TD, we found that overexpression of full-length
Vegfc could partially rescue ccbe1hu3613 mutant lymphatic
phenotypes (supplementary material Fig. S6A,B). However, the shhdriven vegfc expression did not rescue the MO-ccbe1 lymphatic
phenotype and, furthermore, we did not see any rescue of lymphatic
development in MO-ccbe1-injected, hsp70l:GAL4;UAS:vegfc
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. Cooverexpression 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 CCBE1transfected 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.
1245
Development
RESEARCH ARTICLE
RESEARCH ARTICLE
CCBE1 overexpression leads to enhanced levels of
processed, soluble VEGFC in vitro
VEGFC is produced as a secreted precursor protein requiring
proteolytic cleavage of N- and C-terminal propeptides to produce the
highly bioactive mature form, consisting of dimers of the VEGF
homology domain (VHD), which potently activates VEGFR3
signaling (Joukov et al., 1997). Although proteases that can promote
processing of VEGFC have been identified (McColl et al., 2003;
Siegfried et al., 2003), other factors that might regulate the function of
VEGFC in the developing embryo remain to be described. To
determine if the capability of CCBE1 to modulate VEGFC activity in
vivo may be at the level of VEGFC production, release from the cell
surface/ECM or proteolytic processing, we turned to in vitro cell-based
models. 293EBNA-1 cells stably harboring an expression construct
encoding full-length human VEGFC allow analysis of secreted
VEGFC polypeptides in conditioned cell culture medium. These cells
were transfected with an expression vector encoding human CCBE1
(Fig. 5B). In control (empty vector) or mock transfected cells lacking
detectable CCBE1, mature VEGFC was readily detected in
conditioned cell culture media, with partially processed forms detected
at lower levels (Fig. 5C,C′). CCBE1 transfection resulted in a
significant enrichment of all forms of VEGFC in the conditioned cell
culture media but in particular the relative abundance of the mature
form was increased compared with other forms of VEGFC
(Fig. 5C,C′). This indicates that although release of VEGFC from the
cell surface or ECM is enhanced, processing to the mature form is
particularly increased in the presence of CCBE1.
We confirmed the enhanced processing of VEGFC in an
independent cell line, HEK293T cells, using transient transfections of
CCBE1 and VEGFC plasmids; note the increased abundance of
mature VEGFC in the cell culture media in response to CCBE1 in
supplementary material Fig. S7B,C. However, the release of VEGFC
was not generally enhanced in this system as the abundance of other
forms of VEGFC did not increase. In both cell systems, we also
examined the levels of VEGFC present in the cell lysates and found
that in 293EBNA-1 cells we could detect partially processed VEGFC
(i.e. the form containing the N-terminal propeptide and VEGF
homology domain) (supplementary material Fig. S7A) whereas in
HEK293T cells we detected both full-length and partially processed
forms (supplementary material Fig. S7C). The relative levels of
different forms of VEGFC in cell lysates were not markedly altered
in the presence of CCBE1 (supplementary material Fig. S7A,C). We
take this to indicate that the cell-associated VEGFC may be far more
abundant than VEGFC in the medium, hence a small decrease in the
proportion of cell-associated VEGFC, due to release from the cell
surface or ECM, could lead to a significant increase in the relative
abundance of VEGFC detected in the medium on western blots.
Importantly, CCBE1 overexpression had no effect on VEGFC mRNA
levels in vitro or in vivo in zebrafish embryos (Fig. 5D).
In the in vitro assays above, VEGFC and CCBE1 were produced
and secreted from the same cells. To test if CCBE1 needs to be
expressed in the same cell as VEGFC to promote VEGFC processing,
we transfected two different populations of HEK293T cells with
CCBE1 and/or VEGFC and mixed them before detection of VEGFC
in the culture medium. We found that CCBE1 was indeed capable of
enhancing VEGFC processing in trans (supplementary material Fig.
S7B), indicating that CCBE1 can exert its effects on VEGFC outside
the cell. These data, taken together, indicate that in these cell-based
1246
systems CCBE1 regulates VEGFC at a post-transcriptional level, that
this regulation occurs extracellularly and that it is capable of
increasing the release and processing of VEGFC. The degree to which
each of these two mechanisms apply may be dependent on the cell
type, given that the effect on VEGFC release was not observed in the
HEK293T model system.
Constitutively secreted, processed VEGFC rescues ccbe1
loss-of-function phenotypes
To build on these in vitro findings, and given the observation that fulllength Vegfc overexpression cannot rescue the MO-ccbe1 loss-offunction phenotype (supplementary material Fig. S5), we decided to
investigate whether a constitutively secreted, mature form of VEGFC
was sufficient to recue ccbe1 loss of function. To do this, we
generated a truncated form of human VEGFC (ΔNΔCVEGFC),
missing the N- and C-terminal domains but retaining the signal
peptide for secretion, and placed it under the control of the hsp70l
promoter. We used the human form because the proteolytic processing
sites of zebrafish Vegfc are not experimentally validated whereas the
human form has been examined in detail and processing sites
are known (Joukov et al., 1997). We injected DNA for
hsp70l:ΔNΔCVEGFC.t2a.mCherry and generated mosaic embryos.
After a 1-hour heatshock at 26 hpf, embryos injected with
hsp70l:ΔNΔCVEGFC.t2a.mCherry displayed ISV hyperbranching (at
54 hpf, Fig. 6A) as observed previously upon vegfc overexpression
(Fig. 3C; Fig. 5A) and expressed mosaic mCherry. Co-injection of the
hsp70l:ΔNΔCVEGFC.t2a.mCherry with MO-ccbe1 generated a
robust, quantifiable rescue of PL formation in these embryos
compared with MO-ccbe1 controls (Fig. 6A,B). Co-injecting
hsp70l:ΔNΔCVEGFC.t2a.mCherry with MO-vegfr3 did not rescue PL
formation at the horizontal myoseptum by 54 hpf (Fig. 6A,B). Finally,
we found that ISV hyperbranching was present in ccbe1 and vegfr3
morphants injected with hsp70l:ΔNΔCVEGFC.t2a.mCherry
(Fig. 6C), which is consistent with the ability of processed VEGFC to
activate VEGFR2 (KDR – Human Gene Nomenclature Database)
signaling. The ability of a processed form of VEGFC to rescue the
MO-ccbe1 loss-of-function phenotype demonstrates that a deficit in
Vegfc maturation is responsible for venous angiogenesis and
lymphangiogenesis defects in the absence of Ccbe1.
DISCUSSION
Our results show that ccbe1 genetically interacts with vegfc and
vegfr3 and that ccbe1 mutation or depletion suppresses the
formation of excess and abnormal aISV and vISV sprouts driven by
ectopic Vegfc/Vegfr3 signaling. These data highlight the necessity
for functional Ccbe1 for the propagation of (ectopic) Vegfc-driven
signals in the developing embryo. Furthermore, we show that in
ccbe1 and vegfr3 morphants, venous Erk signaling and Vegfr3
activation at Tyr1071/1076 are reduced. Our finding of a block in
Vegfc/Vegfr3 signaling is consistent with phenotypes observed in
humans (Alders et al., 2009) and mice (Bos et al., 2011). Previous
studies have used proximity ligation assays as a readout for Vegfr3
signaling (Bos et al., 2011), and did not observe the reduction of
Vegfr3 signaling in the absence of Ccbe1 that we see here. However,
the previous study was unable to examine the entirety of signaling
in cells at equivalent developmental stages during the induction of
sprouting of lymphatics from the vein. We overcame previous
limitations by utilizing the immunofluorescence visualization of
activated (phosphorylated) Erk in the PCV. We found that during the
induction of secondary sprouting Erk activation is dorsally enriched
and segmentally patterned in the PCV in a Vegfr3-dependent
manner. This assay hence provides improved sensitivity to detect
Development
indicate that partial rescue exclusively in the ccbe1hu3613 mutant
occurs as a result of some retained capability of the ccbe1hu3613
D162E (hypomorphic) mutant protein.
Development (2014) doi:10.1242/dev.100495
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.100495
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 MOccbe1-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.
and predicts the migration route of lymphatic precursors in the trunk
(Hogan et al., 2009a) suggesting that Ccbe1 serves to impart the
necessary post-translational activation of Vegfc (see proposed model
in Fig. 7) in a spatially and temporally regulated manner. Hence, a
migrating lymphatic endothelial cell could be directed through the
embryonic environment by a route pre-determined by the earlier,
local concentration of Ccbe1. To test this idea directly is inherently
difficult, but the hypothesis is supported by overexpression regimes
of ccbe1 and vegfc in an ectopic location, the FP of the neural tube:
here we found that Vegfc-driven sprouting of endothelial cells was
enhanced in the presence of ectopic Ccbe1.
Fig. 7. Ccbe1 activates Vegfc to induce Vegfr3 signaling. Proposed
model for coordination of angiogenesis by Ccbe1, Vegfc and Vegfr3 in the
developing embryo. Vegfc is produced in a largely inactive full-length form
that is processed and released from the cell surface/ECM in a Ccbe1dependent manner to generate the mature, highly active form. Downstream,
arteries respond in a manner dampened by Dll4-dependent suppression of
Vegfr3 signaling (Hogan et al., 2009b), whereas Vegfr3 signaling in veins
induces secondary angiogenesis, which produces both intersegmental veins
and lymphatic vascular precursor cells.
1247
Development
Vegfc-Vegfr3 induced signaling during secondary angiogenesis in
vivo compared with previous approaches. Using this approach and
also cross-reacting antibodies to phospho-Vegfr3, we found a crucial
requirement for Ccbe1 for in vivo Vegfc-Vegfr3-Erk signaling.
Given this function of Ccbe1, its non-autonomous role in zebrafish
(Hogan et al., 2009a) and its extracellular localization (Bos et al.,
2011), we asked if Ccbe1 can directly modulate the levels of bioactive
Vegfc. We did this in cell-based models, and found that upon CCBE1
overexpression, the amount of mature bioactive VEGFC is increased
in the culture medium. CCBE1 can induce higher levels of VEGFC
in trans and hence performs this function outside of the cell in this in
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
the hypochord and dorsal aorta in the midline of the embryonic
trunk. Despite this, secondary sprouts from the vein migrate past this
transcriptional source of ligand on the dorsoventral and mediolateral
axes of the embryo to the horizontal myoseptum and give rise to
PLs. This suggests that spatial presentation of active Vegfc protein
during secondary sprouting must be regulated independently of the
transcriptional source of vegfc. ccbe1 transcript expression precedes
RESEARCH ARTICLE
MATERIALS AND METHODS
Zebrafish strains and transgenesis
Animal work followed guidelines of the animal ethics committees at the
University of Queensland, and the Royal Netherlands Academy of Arts and
Sciences (DEC). Zebrafish transgenic lines Tg(fli1a:EGFP), Tg(−6.5kdrl:mCherry), Tg(−0.8flt1:tdTomato) and Tg(hsp70l:Gal4)1.5kca4 lines were
described previously (Scheer et al., 2001; Lawson and Weinstein, 2002;
Hogan et al., 2009a; Bussmann et al., 2010). N-ethyl-N-nitrosourea (ENU)
mutagenesis was performed as previously described (Wienholds et al., 2002).
The Tg(4xUAS:vegfc) line was generated using a 4xUAS promoter
upstream of the full length zebrafish vegfc cDNA (supplementary material
Fig. S3A) cloned using the Gateway system (Hartley et al., 2000; Walhout
et al., 2000). The Tg(flt4:YFP) reporter line was generated from BAC
DKEY-58G10 as previously described (Bussmann and Schulte-Merker,
2011) and is characterized in detail elsewhere (van Impel et al., 2014). The
Tg(s1173:Gal4) line was kindly provided by Ethan Scott (School of
Biomedical Sciences, University of Queensland). sonic hedgehog (shh)
promoter and floorplate enhancer (Ertzer et al., 2007) was used to drive
Ccbe1 IRES tagRFP or Vegfc IRES mturquoise (supplementary material
Fig. S3C), cloned into the MiniTol2 vector (Balciunas et al., 2006); plasmids
(25 ng/μl) were injected with tol2 transposase mRNA (25 ng/μl) into 1- to
2-cell-stage embryos. All genotyping details and primers are given in
supplementary material Tables S1 and S3. KASPar (KBioscience) was used
for the indicated vegfc alleles.
DNA, RNA and morpholino injections
Constructs
for
transient
DNA
injection
of
the
Tg(hsp70l:ΔΔVEGFC.t2a.mCherry) transgene were generated by PCR
amplifying the N- and C-terminally truncated form of human VEGFC,
containing the endogenous secretion peptide (Joukov et al., 1997) followed by
Gateway recombineering. Primer sequences are given in supplementary
material Table S2. DNA was injected at a concentration of 90 ng/μl at singlecell stage. Morpholinos used are shown in supplementary material Table S4.
The vegfc cDNA used was described previously (Hogan et al., 2009a).
Analysis of ERK and Vegfr3 signaling in zebrafish embryos
Phospho-ERK was detected by immunofluorescence, using the previously
described protocol (Inoue and Wittbrodt, 2011) but modified as follows:
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
serum albumin, 10% goat serum and then with horseradish peroxidaseconjugated anti-goat secondary antibody (1/1000; Cell Signaling, #7074) in
2% blocking solution (Roche) dissolved in 100 mM maleic acid with 150
mM NaCl (pH 7.4). Phospho-ERK was subsequently detected using the
TSA reagent (Perkin Elmer) as per manufacturer’s guidelines. As a control
for antibody specificity, embryos were incubated in 100 μM PD98059 (Cell
Signaling) from 24 hpf. Embryos were vibratome sectioned (100 μm) (Leica
VT100S) and imaged as described below.
Zebrafish phospho-Vegfr3 was detected following immunoprecipitation
(IP) with anti phospho-VEGFR3 (Cell Applications, CB5793) using the
same antibody. One hundred and fifty embryos were lysed overnight at 4°C
in modified RIPA buffer containing 1 mM EDTA, 4% protease inhibitor
(Sigma), 1% phenylmethylsulfonyl fluoride (PMSF; Sigma) and 1% Halt
phosphatase inhibitor cocktail (Thermo Fisher Scientific). IP was carried out
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
Scientific). Standard western blotting approaches were used to detect Vegfr3
1248
with both anti-phospho-VEGFR3 (1/1000; Cell Applications, CB5793) and
anti (non-phosphorylated) VEGFR3 (1/1000; Cell Applications, CB5792).
Mouse anti-myosin light chain 1 and 3f (1/100; Developmental Studies
Hybridoma Bank, F310) was used to detect a loading control band.
Quantification relative to IgG light chain (phospho-Vegfr3 only) or Myosin
used ImageJ across three biological replicates. Only phospho-Vegfr3 was
assessed and not total Vegfr3, owing to a limitation of the cross-reacting
antibodies in only detecting bands post-IP for phospho-Vegfr3.
VEGFC processing/secretion in vitro
For analysis of VEGFC processing and secretion two approaches were used.
Briefly, using 293EBNA-1 cells stably expressing a full-length form of
human VEGFC (Karnezis et al., 2012) were transiently transfected
[Lipofectamine 2000 (Invitrogen)] with a CCBE1 expression vector after a
medium change to a serum-free chemically defined medium (Pro293aCDM, Lonza). Both supernatant and cell lysates were subsequently collected
for western blotting. 293EBNA-1 cells were maintained in Dulbecco’s
Modified Eagle Medium, supplemented with 10% fetal bovine serum,
50 mM L-glutamine, 50 μg/ml penicillin, 50 μg/ml streptomycin and
100 μg/ml hygromycin B (Roche Diagnostics). Cells were lysed in 0.1%
SDS, 50 mM Tris, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate,
pH 8.0, 1 mM PMSF, 10 μg/ml aprotinin and 10 μg/ml leupeptin. Lysates
were incubated at 4°C for 20 minutes with gentle agitation and centrifuged
at 13,000 g for 10 minutes. Western blotting was performed with antibodies
targeting VEGFC (R&D Systems, BAF752) or CCBE1 (Abcam, ab101967).
For cell mixing experiments, separate populations of HEK293T cells were
transfected with constructs expressing CCBE1, VEGFC or both. Cells were
passaged and mixed as indicated after 24 hours. From mixed cultures,
conditioned supernatants were collected and cells were lysed in RIPA/SDS
buffer after another 48 hours. Supernatant and cell lysates were isolated and
western blotting analysis performed using VEGFC (VEGFC isoform 103
antibody, antibodies-online) and CCBE1 (HPA041374, Sigma) antibodies.
qPCR detection of Vegfc and Ccbe1 expression
For expression analysis in the cultured cells, RNA was isolated using
RNeasy Mini Kit (Qiagen), and cDNA synthesized using High Capacity
cDNA Reverse Transcription Kit (Applied Biosystems). For expression
analysis, Taqman Gene Expression Assays were employed [VEGFC
(Hs01099203_m1), GAPDH (Mm99999915_g1)] using Taqman Fast
Universal PCR Master Mix (2×) (Applied Biosystems) as per
manufacturer’s instructions and analyzed on a 7300 RT-PCR machine
(Applied Biosystems). For zebrafish qPCR, RNA was extracted at 24 hpf
from 20 zebrafish embryos. cDNA was synthesized using the Superscript III
Kit (Invitrogen) and reactions used SYBR Green kit (Applied Biosystems)
analyzed on a 7500 RT-PCR machine (Applied Biosystems). Data were
normalized using the geometric average of ef1α (eef1a1l1), rpl13 and rps29,
which were found to be the most stable reference genes using GeNorm with
data displayed as arbitrary units (A.U.).
Imaging
Confocal imaging was performed on live embryos mounted laterally using
a Zeiss 510 or a Leica SPE confocal microscope at the indicated stages.
Sections were imaged using a Zeiss 710 FCS confocal microscope.
Statistical analysis and mutation effect prediction
For genetic interaction data, χ2 tests were performed using GraphPad
(http://graphpad.com/quickcalcs/chisquared1.cfm). PolyPhen-2 was used to
evaluate mutation pathogenicity (http://genetics.bwh.harvard.edu/pph2/).
Acknowledgements
We thank C. Neyt, G. van de Hoek, N. Chrispijn and M. Witte for technical
assistance; Dagmar Wilhelm for advice; Nathan Lawson for providing an initial PErk immunofluorescence protocol; and Ethan Scott for providing the Tg(s1173:Gal4)
line. Imaging was performed in the Australian Cancer Research Foundation’s
Dynamic Imaging Facility at IMB and at the Hubrecht Imaging Center (HIC).
Competing interests
The authors declare no competing financial interests.
Development
CCBE1 mutations have been shown to be responsible for
generalized lymph vessel dysplasia in humans (Alders et al., 2009;
Connell et al., 2010). Our finding that Ccbe1 is a crucial modulator
of the Vegfc/Vegfr3 pathway during embryo development, suggests
that a deficiency of VEGFR3 signaling may be responsible for the
lymphatic aspects of Hennekam Syndrome. Other aspects of this
syndrome that are distinct from Milroy’s disease might be due to
other, yet to be identified, functions of CCBE1.
Development (2014) doi:10.1242/dev.100495
Author contributions
L.L.G. designed and performed experiments, analyzed data and co-wrote the
manuscript; T.K., D.S., N.C.H., K.K., G.R., N.I.B. and A.v.I. designed and
performed experiments, analyzed data and edited the manuscript; S.A.S. and
M.G.A. designed experiments, analyzed data and edited the manuscript; S.S.-M.
and B.M.H. designed experiments, analyzed data and co-wrote the manuscript.
Funding
This work was supported by an Australian Research Council Future Fellowship
[FT100100165 to B.M.H.]; a European Molecular Biology Organization Long-Term
Fellowship [LTRF 52-2007 to T.K.]; Veni grants from The Netherlands Organisation
for Scientific Research (NWO) [916.10.132 to T.K.; 863.11.022 to G.R.]; Marie
Curie Intra-European Fellowships (to D.S. and A.v.I.); National Health and Medical
Research Council of Australia (NHMRC) project grants [631657 and
APP1050138]; a Program Grant [487900 to M.G.A. and S.A.S.] and Research
Fellowships (to M.G.A. and S.A.S.) from the National Health and Medical
Research Council; and KNAW (S.S.-M.).
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.100495/-/DC1
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Development
RESEARCH ARTICLE
Fig. S1. Morphology of vegfc, ccbe1 and vegfr3 mutants and additional phenotypes of vegfc mutants. (A-D) Mutant
hu5055 that specifically lacks lymphatic vasculature (Figure 2F and 2H) (D) is undistinguishable from vegfr3 (C) and ccbe1
(B) mutants. (E-H) Gross morphology of vegfc mutants. Confocal projections in the Tg(fli1a:EGFP) background. At 5 dpf,
vegfchu6124 homozygote mutant display a specific loss of the lymphatic vasculature with apparently normal blood vasculature
(F), as compared to wildtype (E). Mutant embryos that survive after 5dpf, display severe edema in the trunk area, as well
as around the eyes (H) compared with wildtype siblings (G). (I-K) Phenotypes of heterozygous and homozygous vegfc
mutants. (I) Angiographies in Tg(fli1a:EGFP) transgenic wildtype sibling, vegfchu6410 heterozygote and homozygote mutant
embryos reveal the presence of blood flow and thoracic duct (TD) defects in mutants. In the vegfchu6410 early stop allele, heterozygote mutant embryos display a partial loss of TD development while in homozygotes no TD is observed. (J) Secondary
sprouting in plcg1 morphants. In plcg morphants, arteries are missing facilitating the visualization of venous sprouts. As
compared to wildtype, only a few secondary sprouts are observed in heterozygote mutants, and even fewer in homozygote
mutant embryos. (K) PLs in a wildtype sibling, vegfchu6410 heterozygote and homozygote mutant embryos. As compared to
wildtype, heterozygote embryos develop less PLs. In homozygote vegfchu6410 mutants no PLs are present.
Fig. S2. Additional controls for vegfc RNA and MO-dll4 injections in wildtype and ccbe1 mutants. In wildtype embryos as well as in ccbe1 mutants, the single injection of MO-dll4 produces no phenotype at 30 hpf. As previously reported
(Hogan et al, 2009) vegfc RNA injections in wildtype produce only a weak phenotype in the ISVs, that is not observed in
ccbe1 mutants.
Fig. S3. Validation of the Tg(UAS:vegfc), Tg(shh:ccbe1) and Tg(shh:vegfc) transgenic line. (A) The Tg(4xUAS:vegfc) line, was crossed to the Tg(hsp70l:Gal4)(Scheer et al, 2001) to provide inducible ubiquitous expression of vegfc after
heat-shock, or to the Tg(s1173:Gal4)(Scott & Baier, 2009) to provide specific expression in the tectal cell bodies, hindbrain,
strong ocular muscles, and eye: bipolar cells. Schematic representation of the transgenic constructs. (B) In situ hybridization
(as described (Schulte-Merker, 2002)) at 24hpf in a clutch of incrossed Tg(hsp70l:Gal4;4xUAS:vegfc) embryos. n=32/83
embryos displayed ectopic vegfc expression with an expected frequency of 50%. In a clutch of incrossed Tg(s1173:Gal4;4xUAS:vegfc), 22/68 embryos displayed ectopic vegfc expression with an expected frequency of 50%. (C) Schematic representation of the shh:ccbe1 and shh:vegfc contructs. We generated these constructs using the MiniTol2 vector(Balciunas
et al, 2006) to specifically overexpress either of these genes in the FP using the shh promoter and a FP specific enhancer.
The effective expression of these genes can be monitored due to the integration of an IRES tagRFP or IRES mturquoise
reporter. (D) Confocal projections in the Tg(fli1a:EGFP) background. At 56hpf in Tg(shh:ccbe1) embryos, RFP fluorescence
is detected in the floorplate (FP).
Fig. S4. VEGFR3 conservation across species. Sequence conservation with zebrafish in the region of the immunizing
peptide for the Phospho-VEGFR3 antibody (Cell Application-CB5793). Strong conservation across species is observed in
this kinase domain with the percentage identity of the human protein to mouse 97%, and to zebrafish is 93%. The phosphorylated tyrosine residues are indicated by arrows.
Fig. S5. Antibody specificity control: cross-reacting VEGFR3 antibodies detect the same bands and reduction
in morphants. Confirmed of the specificity of the band detected with the Phospho-VEGFR3 antibody, using a VEGFR3
antibody designed to recognise the cytoplasmic domain (Cell Application-CB5792). After a preliminary IP with the Phospho-Vegfr3, we performed IB with the second VEGFR3 antbody. Bands of the same size were observed and identical reductions in (Phospho-)Vegfr3 observed as using the P-VEGFR3 antibody.
Fig. S6. Tg(shh:vegfc) partly rescues lymphatic development in ccbe1hu3613 mutants but not in ccbe1 morphants.
(A) Confocal projections in the Tg(flt4:YFP) background. In wildtype embryos PLs are visible at 3.5dpf (arrows) and TD is
present at 5dpf (arrow heads), but not in ccbe1hu3613 mutants, PLs and TD are missing (asterisks). However upon vegfc overexpression in the Tg(shh:vegfc) some PLs (arrows) and TD fragments (arrow heads) are observed in ccbe1hu3613 mutants.
(B) Quantitation of PLs and TD rescue in the Tg(shh:vegfc) in the absence of ccbe1. When vegfc is ectopically expressed in
the floorplate, more than 70% of ccbe1hu3613 mutants have PLs, and 55% develop TD fragments. (C) Confocal projections in
the Tg(flt4:YFP) background. In wildtype embryos PLs are visible at 3.5dpf (arrows), but not in ccbe1 morphants (asterisks).
Upon vegfc overexpression in the Tg(shh:vegfc) PLs (asterisks) are not rescued in ccbe1 morphants. (D) Quantitation of
PLs rescue in the Tg(shh:vegfc) in wildtypes and ccbe1 morphants. When vegfc is ectopically expressed in the floorplate,
0% of ccbe1 morphants have PLs. (E) In the Tg(hsp70l:Gal4;4xUAS:vegfc) line no PL or TD rescue was observed after
ccbe1 morpholino injection.
Fig. S7. CCBE1 enhances processing of VEGFC in a non-cell-autonomous manner. (A) Control for Figure 5B and C.
Western blot of 293EBNA-1 cells (stably expressing VEGFC) shows no change in the levels of a partially processed form
of VEGFC (containing the N-terminal propeptide and VEGF homology domain) in the cell lysates when CCBE1 plasmid
is transfected. Full-length VEGFC is masked by a non-specific band as indicated; this was demonstrated by comparison
with 293EBNA-1 cells that had not been stably transfected to express full-length VEGFC (not shown). (B) HEK293T cells
transfected with VEGFC secrete into the medium predominantly the 31 kDa form when they are mixed (in a 1:1 ratio) with
untransfected control cells (lane 1). When co-transfected with CCBE1 in the same cell population CCBE1 enhances processing of VEGFC leading to an increase of the mature 21 kDa form (3rd lane). Similarly, mixing cells in which CCBE1 is
transfected in one cell population and VEGFC separately transfected into another, leads to an increase of the mature 21
kDa form (2nd lane) indicating that CCBE1 can induce processing of VEGFC in a non-cell-autonomous fashion. The top
blot shows detection of CCBE1 in the cell lysate, and the bottom blot shows detection of VEGFC in conditioned media. (C)
Western blot comparing HEK-293T cell lysates and medium. When cells are transfected with VEGFC both the full-length
and the partially processed 31 kDa forms are detected in the cell lysate (left blot, lane 1). When cotransfected with CCBE1
and VEGFC both the full-length and partially processed 31 kDa forms are detected in the cell lysate, with no marked differences. As detected in the medium in the same experiment, CCBE1 enhances processing of VEGFC leading to the formation
of the mature 21 kDa form (right blot). PCS2 denotes the expression vector lacking DNA encoding VEGFC or CCBE1.
Supplementary references
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(2006) Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genetics 2: e169
Hogan BM, Herpers R, Witte M, Helotera H, Alitalo K, Duckers HJ, Schulte-Merker S (2009) Vegfc/Flt4 signalling is suppressed
by Dll4 in developing zebrafish intersegmental arteries. Development 136: 4001-4009
Scheer N, Groth A, Hans S, Campos-Ortega JA (2001) An instructive function for Notch in promoting gliogenesis in the zebrafish
retina. Development 128: 1099-1107
Schulte-Merker S (2002) Looking at embryos. In Zebrafish: A practical approach, Nüsslein-Volhard CaD, R. (ed), pp 39-58.
New York: Oxford University Press
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Table S1. Primers used to monitor transgene expression by PCR
Transgenic line
shh:ccbe1 IRES RFP
Primer sequence
For: 5’-AGCAACACCGAGATCAATAAGCAGA-3’
Rev: 5’-AGGGCCCTTCGTATGATTGAGTGCT-3’
shh:vegfc IRES mturquoise
For: 5’-GCGTTCAGCGGGTAGTGTGGATG-3’
Rev: 5’-TGGGAAATCCTCAAATGAAGAATGT-3’
1
Table S2. Primers used to clone ∆∆VEGFC into the gateway middle entry
vector
∆∆VEGFC
For1: 5’-GGGG-ACA AGT TTG TAC AAA
AAA GCA GGC T-ACC-ATG CAC TTG CTG
GGC TTC TTC TCT GTG GCG TGT TCT
CTG CTC GCC GCT GCG-3’
For2: 5’-TGT TCT CTG CTC GCC GCT GCG
CTG CTC CCG GGT CCT CGC GAG GCG
CCC GCC GCC GCC GCC GCC GCA CAT
TAT AAT ACA GAG ATC-3’
Rev: 5’-GGGG-AC CAC TTT GTA CAA CAA
AGC TGG GT- TTA ACG TCT AAT AAT
GGA ATG AAC
2
Table S3. Primers used for genotyping assays
Mutant Line
ccbe1hu3613
Primer sequence
For1: 5’-GAAGCGATTCATGCCGCCCAG-3’
Rev1: 5’-CTCTCTCGCCCTTGGTGCATG-3’
For2: 5’-CATTGATGAATGTGCCAACAA-3’
Rev2: 5’-TCTCGCCCTTGGTGCATGTCT-3’
vegfchu5055
For: 5’-ACTGAAAGGAAATAACTTTTG-3’
Rev: 5’-GTCCAGTCTTCCCCAGTATGT-3’
vegfr3hu4602
For: 5’-TGATCTTGATTTCTGTATGTATTTCAGTGAA-3’
Rev: 5’-TAGAGATCTTTGTTGATCTTG-3’
vegfchu6410
ALT: 5’-GAAGGTGACCAAGTTCATGCTGGCTGCTTTCATCAATCTTGAACTTTT-3’
ALA: 5’-GAAGGTCGGAGTCAACGGATTGGCTGCTTTCATCAATCTTGAACTTTA-3’
C2: 5’-GGCGGTTCTAAATTAATAGTCACTCACTT-3’
vegfchu5142
ALT: 5’-GAAGGTGACCAAGTTCATGCTAAGAGTTTGGGGCTACAAACACCTT-3’
ALG: 5’-GAAGGTCGGAGTCAACGGATTGAGTTTGGGGCTACAAACACCTG-3’
C2: 5’-AGACAGACACGCAGGGTGGTTTATA-3’
vegfchu6124
ALT: 5’-GAAGGTGACCAAGTTCATGCTAACCGCACCAAATGTGTGTGTGAAT-3’
ALA: 5’-GAAGGTCGGAGTCAACGGATTAACCGCACCAAATGTGTGTGTGAAA-3’
C2: 5’-CCKTCTCCCTTTTAAGAAGCACTTGTT-3’
vegfchu5055
ALT: 5’-GAAGGTGACCAAGTTCATGCTCAGCTGTGTCCGGCCTCCAT-3’
ALC: 5’-GAAGGTCGGAGTCAACGGATTAGCTGTGTCCGGCCTCCAC-3’
C2: 5’-CCKTCTCCCTTTTAAGAAGCACTTGTT-3’
3
Table S4. Morpholino sequences
Morpholino name
ccbe1 ATG MO
vegfc ATG MO
vegfr3 ATG MO
dll4 Spl MO
Sequence
5’-CGGGTAGATCATTTCAGACACTCTG-3’
5’-GAAAATCCAAATAAGTGCATTTTAG-3’
5’- CTCTTCATTTCCAGGTTTCAAGTCC-3’
5’-TGATCTCTGATTGCTTACGTTCTTC-3’
4