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VASCULAR BIOLOGY
Role of synectin in lymphatic development in zebrafish and frogs
*Karlien Hermans,1,2 *Filip Claes,1,2 Wouter Vandevelde,1,2 Wei Zheng,3 Ilse Geudens,1,2 Fabrizio Orsenigo,4
Frederik De Smet,1,2 Evisa Gjini,5 Kristof Anthonis,1,2 Bin Ren,6 Dontcho Kerjaschki,7 Monica Autiero,1,2 Annelii Ny,1,2
Michael Simons,6 Mieke Dewerchin,1,2 Stefan Schulte-Merker,5 Elisabetta Dejana,4,8 Kari Alitalo,3 and Peter Carmeliet1,2
1Vesalius
Research Center, Vlaams Iustituut voor Biotechnologie (VIB), Leuven, Belgium; 2Vesalius Research Center, Katholieke Universiteit Leuven,
Leuven, Belgium; 3Molecular Cancer Biology Program, Biomedicum Helsinki, Haartman Institute, University of Helsinki, Helsinki, Finland; 4IFOM, FIRC
Institute of Molecular Oncology, Milan, Italy; 5Hubrecht Institute-KNAW and UMC, Utrecht, The Netherlands; 6Section of Cardiovascular Medicine, Yale
University School of Medicine, New Haven, CT; 7Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria; and 8Department of
Biomolecular Sciences and Biotechnologies, University of Milan, Milan, Italy
The molecular basis of lymphangiogenesis remains incompletely characterized. Here, we document a novel role for
the PDZ domain-containing scaffold protein synectin in lymphangiogenesis using genetic studies in zebrafish and
tadpoles. In zebrafish, the thoracic duct
arises from parachordal lymphangioblast cells, which in turn derive from
secondary lymphangiogenic sprouts
from the posterior cardinal vein. Morpholino knockdown of synectin in zebrafish
impaired formation of the thoracic duct,
due to selective defects in lymphangiogenic but not angiogenic sprouting. Synectin genetically interacted with Vegfr3
and neuropilin-2a in regulating lymphangiogenesis. Silencing of synectin
in tadpoles caused lymphatic defects
due to an underdevelopment and im-
paired migration of Prox-1ⴙ lymphatic
endothelial cells. Molecular analysis further revealed that synectin regulated
Sox18-induced expression of Prox-1 and
vascular endothelial growth factor C–
induced migration of lymphatic endothelial cells in vitro. These findings reveal a
novel role for synectin in lymphatic
development. (Blood. 2010;116(17):
3356-3366)
Introduction
The lymphatic vasculature regulates interstitial fluid homeostasis, fat resorption, immune defense, inflammation and cancer
metastasis. The molecular basis of the lymphatic development
remains incompletely understood. 1 Lymphatic endothelial
cells (LECs) derive from venous blood endothelial cells
(BECs).2-5 Sox186 and Prox-1,2-5 as well as miRNAs,7 play a
key role in this process. Intriguingly, despite their venous
origin, formation of lymphatics relies in part on signals that
participate in arterial development. For instance, the forkhead
transcription factors Foxc1/2 are required for arterial specification and sprouting of LECs.8,9 In addition, the PDZ interaction
site in EphrinB2, itself a marker of arterial endothelial cells
(ECs), is essential for lymphatic development.10 Similarly,
Dll4/Notch signaling regulates arterial and lymphatic
development.11
Recently, we identified the PDZ domain–containing scaffold
protein synectin (GIPC1) as a regulator of arterial but not venous
growth,12 at least in part through control of Vegfa signaling.13
Prompted by the finding that similar factors control arterial and
lymphatic development, we studied whether synectin also regulated lymphatic development. Although PDZ domain–dependent
signaling is crucial for lymphatic development,10 a role for synectin
in lymphatic development has not been documented yet. We
therefore explored a role of synectin in this process using zebrafish
and Xenopus tadpole models.
Methods
Zebrafish analysis
Fli1:eGFPy1,14 Gata-1:DsRed15 zebrafish and PLC␥y10 16 zebrafish were
maintained under standard conditions. All morpholinos were previously
reported and purchased from Gene Tools (supplemental Table 1;
available on the Blood Web site; see the Supplemental Materials link at
the top of the online article). Different doses of morpholinos were
injected into single- to 4-cell stage embryos, as previously described.12
Phenotyping is described in supplemental material. All animal experimentation was approved by the Katholieke Universiteit Leuven institutional ethical committee.
Xenopus analysis
The generation and characterization of transgenic Flk1:eGFP Xenopus
laevis frogs will be reported elsewhere (manuscript in preparation). Eggs
were obtained by natural mating of hormonally induced Flk1:eGFP
females and wild-type males and injected with different doses of synectinspecific or control morpholinos (Gene Tools, supplemental Table 1) into the
2-cell stage.17 Nonoverlapping translational blocking synectin morpholinos
were designed based on published GenBank Xenopus laevis sequences
(NM_001088594) and morpholino efficiency was tested using an in vitro
luciferase reporter assay17 and immunoblotting (supplemental Methods).
Tadpoles were kept in tadpole growth medium (0.1 ⫻ MMR) at 18°C until
gastrulation was completed and from then on at 22°C.17 Phenotyping is
described in supplemental Methods.
Submitted November 16, 2009; accepted June 30, 2010. Prepublished
online as Blood First Edition paper, July 14, 2010; DOI 10.1182/blood-200911-254557.
The online version of this article contains a data supplement.
*K.H. and F.C. contributed equally to this work.
© 2010 by The American Society of Hematology
3356
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
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BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
Immunohistochemistry and in situ hybridization
SYNECTIN IN LYMPHATIC DEVELOPMENT
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Fli1:eGFPy1 zebrafish embryos were fixed overnight in 4% paraformaldehyde/PBS at 4°C and processed for paraffin- or cryosectioning. Sections
(7 ␮m) were stained using a polyclonal chicken anti–human synectin
antibody (Abcam) and a rabbit anti-GFP antibody (Torrey-Pines Biolabs),
which were then detected using biotinylated anti-chicken (Abcam) and
Alexa Fluor 488–conjugated anti-rabbit secondary antibodies, respectively.
Sections were mounted with Vectashield DAPI (4⬘,6-diamidino-2phenylindole; Vector Laboratories).
For Xenopus in situ hybridizations, embryos were fixed in Memfa
fixative and whole-mount in situ hybridization using antisense probes for
Prox-117 and synectin (primer sequence, supplemental Table 1) was
performed as described.17 Analysis of Prox-1–stained areas in the tadpole
tail was performed as reported (supplemental Methods).17 For zebrafish
whole-mount in situ hybridization, dechorionated embryos were fixed
overnight in 4% paraformaldehyde at 4°C. In situ hybridization was
performed as described,12 using antisense probes for EphrinB2a,12 Gridlock
(Grl),18 EphB4,19 Flt4,12 and Tie-2.20 Both sections and whole-mounts were
visualized on a Zeiss Axioplan 2 imaging microscope.
their location between the DA and PCV to fuse and establish the
TD (3–6 days postfertilization [dpf]).
We and others previously reported that synectin is expressed
in the PCV when secondary lymphangiogenic sprouts form,12,26
but did not characterize synectin expression in lymphatic
vessels. When using Fli1:eGFPy1 embryos, which express
enhanced green fluorescent protein (eGFP) in blood and lymph
vessels,14,25 we found that synectin was expressed in the DA,
PCV, PL cells, and TD (Figure 1A-F) by double immunostaining
for GFP and synectin in 3- and 7-dpf embryos. Furthermore, in
agreement with previous findings,12,26 synectin was detectable in
the head (not shown) and in the neural tube, pronephric duct,
somites, and gut (Figure 1A-F). Widespread expression of
synectin has been observed in human, mouse, and Xenopus
tissues.27-29 Of note, synectin was also detectable in primary and
immortalized human LECs (supplemental Figure 2), consistent
with previous observations.30
Cell culture experiments
Incomplete silencing of synectin in zebrafish does not
affect angiogenesis
Human microvascular lung endothelial cells (HMVEC-Lly; Lonza,
Invitrogen) and adult dermal LECs (HMVEC-dLyAd; Lonza, Invitrogen),
telomerase transfected dermal LECs (hTERT-HDLECs21), immortalized (i)
LECs, and human umbilical vein ECs (Lonza, Invitrogen) were grown in
endothelial growth medium (EGM)-2–MV medium (Lonza, Invitrogen) at
37°C. Human dermal LEC (HDLEC) cells were obtained from Promocell
and cultured in endothelial medium provided by the supplier. Synectin
expression was evaluated by quantitative reverse-transcription polymerase
chain reaction (qRT-PCR; supplemental Table 1 primer sequences). The
LEC spheroid and Sox18-mediated lymphatic reprogramming assays are
described in supplemental Methods.
Statistical analysis
Absolute values were used to calculate mean ⫾ SEM. Significance levels
were calculated by unpaired Student t test, univariate, or multivariate
analysis with the treatment groups as fixed factor and experiment as
covariate. To determine the penetrance of the zebrafish and tadpole
phenotypes, we counted the number of embryos, exhibiting the (different
severities of) morphant phenotype. We used ␹2 analysis to determine
whether the severity distribution differed between treatment groups.
Results
Synectin is expressed in LECs in zebrafish
Because the expression of synectin in lymphatics has not been
documented yet, we analyzed its expression pattern during lymphangiogenesis in zebrafish embryos. For reasons of clarity, we
will briefly explain first how lymphatics develop in zebrafish
(supplemental Figure 1).11,20,22-25 The thoracic duct (TD) is the first
perfused lymphatic, located between the dorsal aorta (DA) and
posterior cardinal vein (PCV). TD development initiates between
30 and 50 hours postfertilization (hpf) when secondary sprouts
branch off dorsally from the PCV. Half of these sprouts are
angiogenic, as they connect to primary (arterial) intersomitic
vessels (aISVs), which thereby become venous ISVs (vISVs). The
other half of these secondary sprouts are lymphangiogenic, as they
migrate dorsally to the horizontal myoseptum, where they form (by
36-60 hpf) a transient, nonlumenized string of parachordal lymphangioblast (PL) cells, termed so because they are precursors of
LECs forming the TD. Beyond 60 hpf, PL cells navigate ventrally
and dorsally alongside aISVs, with ventral sprouts migrating to
To study the role of synectin in lymphangiogenesis, we silenced
its expression (synectinKD) in Fli1:eGFPy1 zebrafish embryos
using previously characterized SynATG1 and SynATG2 morpholinos.12 As synectin regulates angiogenesis, we first determined a
range of submaximal morpholino doses, which only minimally
affected blood vessel development, to avoid that angiogenic
defects secondarily caused lymphatic defects. At a dose of 9 ng
SynATG1 and 6 ng SynATG2 per embryo, only 7% of synectinKD
embryos displayed subtle vascular defects, including a slightly
thinner DA and a few malformed ISVs (Figure 2A-B and
supplemental Table 2). The remainder of the blood vasculature
in the head, trunk, tail and subintestinal vessels developed
normally, had a normal size and shape, and exhibited comparable branching and density (supplemental Figure 3). Blood flow
in large axial vessels and ISVs was normal in 2-dpf synectinKD
Gata-1:DsRed embryos, harboring DsRed⫹ erythrocytes15 (Figure 2C-D). Furthermore, arterio-venous differentiation of large
axial vessels and primary ISVs was normal in synectinKD
embryos, as evidenced by in situ hybridization for arterial and
venous markers at 28 hpf (Figure 2E-L). Only morphant
embryos with normal trunk circulation and body size and
without developmental delay, tissue malformations, edema, or
toxic signs were analyzed. (We refer to synectinKD embryos
without mentioning that silencing was incomplete.)
Silencing of synectin in zebrafish impairs TD formation
We studied TD formation by measuring its length at 7 dpf, when
this vessel was completely formed in control embryos, using
previous methods.11 Briefly, the TD length was measured in
10 somites and expressed as percentage of this trunk fragment
(supplemental Figure 4).11 Because the penetrance of the lymphatic
phenotype was variable (supplemental Note 1), we counted the
fraction of embryos with severe, intermediate, or subtle lymphatic
defects. For SynATG1 and SynATG2, the severity and penetrance of
the knockdown phenotypes were dose-dependent (supplemental
Tables 3-4); for reasons of brevity, only the most penetrant
phenotype is shown (9 ng of SynATG1). In addition, lymphatic
development in zebrafish occurs in a metameric pattern, since
lymphangiogenic sprouts, and hence the PL and TD, form,
statistically, at every second unilateral somite segment, giving rise
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3358
HERMANS et al
BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
Figure 1. Synectin is expressed in lymphatic vessels in zebrafish embryos. Scale bars represent 10 ␮m in panels A-F. In all panels, the dorsal side of the embryo is
at the top of the figure. G indicates gut; NT, neural tube; P, pronephric duct; and S, somite. (A-C) Transverse sections through the trunk of a 3-dpf wild-type Fli1:eGFPy1
zebrafish embryo, stained by immunohistochemistry using a polyclonal anti–human synectin antibody, and by DAPI nuclear stain, revealing prominent synectin
expression in the NT, G, S, dorsal aorta (DA, white arrow), posterior cardinal vein (PCV, green arrow) and parachordal lymphangioblast (PL) cells (white arrowhead).
Insets show magnification of the boxed areas (PL cells) in each panel. (D-F) Using the same staining procedure on transverse sections through the rostral trunk of a 7
dpf wild-type Fli1:eGFPy1 zebrafish embryo, similar widespread synectin expression in the NT, G, S, P, DA (white arrow), PCV (green arrow), and thoracic duct (white
arrowhead) was observed.
to discontinuous TD fragments that fuse with each other to
establish a continuous perfused TD (supplemental Figure 1).11
In control embryos, injected with a 5-base-pair mismatch
morpholino (SynCtr), the TD developed as a continuous lymphatic
by 7 dpf (Figure 3A,C,E and supplemental Table 3). In contrast,
32% of synectinKD embryos completely lacked a TD by 7 dpf
(“severe” defects; Figure 3B,D-E and supplemental Table 3).
Follow-up studies further revealed that TD development was not
rescued at 10 dpf (supplemental Figure 3E-F), indicating that
lymphatic formation was aborted and not merely delayed in these
synectinKD embryos. In another 14% of morphants, TD rudiments
developed over only 1 to 3 somites into an incomplete string of
disconnected segments (“intermediate” defects; Figure 3E and
supplemental Table 3). Incomplete TD formation over 30%-90% of
its normal length was further observed in 36% of synectinKD
embryos (“subtle” defects; Figure 3E and supplemental Table 3),
while TD development was normal in the remainder of the
morphant embryos. Because the TD develops in a metameric
pattern (see above), the finding that the TD formed over for
instance only 20% of its length implies that TD development was
aborted in the 8 other of the 10 somites analyzed. Thus, silencing of
synectin aborted lymphatic development completely in all somites
in a third of all morphants, and partially in a fraction of the somites
in another 50% of the morphants.
Knockdown of synectin impairs early lymphatic
development in zebrafish
We then explored whether the TD defect in synectinKD morphants
was related to a defect in PL formation by quantifying its length in
10 somites in Fli1:eGFPy1 embryos at 60 hpf (using a similar
method as used for the TD). In control embryos, PL cells were
detected in nearly every somite segment (Figure 4A,C and supplemental Table 4). By contrast, the PL was absent in 28% of
synectinKD embryos (Figure 4B-C and supplemental Table 4) or
formed over only 10%-30% of its normal length in another 27% of
morphants (Figure 4C and supplemental Table 4). An additional
40% of synectinKD embryos displayed PL cells in only 3 to
9 somites, while in the remaining 5%, the PL string developed
normally. The close correlation between TD and PL defects
suggests that silencing of synectin impaired early lymphatic
formation.
Knockdown of synectin impairs secondary sprout formation
We then evaluated whether absence of PL cells resulted from
lymphangiogenic sprout defects. In case lymphangiogenic but
not angiogenic sprout formation would be defective, we expected that 50% of all secondary sprouts would be affected/
absent. Several assays were used to address this issue. First, we
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BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
SYNECTIN IN LYMPHATIC DEVELOPMENT
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Figure 2. Normal blood vessel development in synectinKD embryos. In all panels, head faces left and dorsal is up. Scale bars represent 50 ␮m in panels A-B
and E-L, and 150 ␮m in panels C-D. DA indicates dorsal aorta; DLAV, dorsal longitudinal anastomosing vessel; and PCV, posterior cardinal vein.
(A-B) Confocal side views of the trunk of 48-hpf control (A) and synectinKD (B) Fli1:eGFPy1 zebrafish embryos, showing normal vascular morphogenesis in the morphant
embryo. Large arrow indicates the DLAV while the small arrow indicates an intersegmental vessel. (C-D) Macroscopic side views of 48-hpf control (C) and synectinKD
(D) Gata-1:DsRed embryos expressing DsRed specifically in erythrocytes. SynectinKD embryos show a normal distribution of erythrocytes throughout the body,
indicating a fully functional circulation. (E-L) Bright-field side views of the trunk of 28-hpf control (E-F,I-J) and synectinKD (G-H,K-L) embryos stained by whole-mount in
situ hybridization for a panel of arterial (Grl, E,G; EphrinB2a, F,H) and venous markers (Flt4, I,K; EphB4, J,L). No difference in expression of any of these markers could
be observed in the synectinKD embryos, indicating that arterio-venous differentiation had occurred normally in these embryos. Red and blue lines denote DA and PCV,
respectively.
used whole-mount in situ staining for Tie-2 to identify both
angiogenic and lymphangiogenic secondary sprouts at 48 hpf. In
81% of control embryos (n ⫽ 66), 8 to 10 secondary sprouts
developed in the 10 unilateral somites analyzed, (ie, in nearly
every somite [Figure 4D-D⬘]); in another 17%, 6 to 8 secondary
sprouts developed, while very rarely (in 2%), only half the
number of secondary (4 to 5) sprouts formed. By contrast, in
22% of synectinKD embryos, only half of the normal number of
secondary sprouts formed (n ⫽ 74; P ⬍ .001; Figure 4E-E⬘),
while in another 41%, only 6 to 8 Tie-2⫹ sprouts developed
(N ⫽ 74; P ⬍ .001 vs control), while the remainder fraction
displayed no secondary sprouting defects. Notably, comparable
fractions of morphant embryos suffered severe secondary sprout
defects, and lacked a PL and TD, suggesting that the latter and
former processes were linked.
Second, we used the genetic PLC␥1y10 model to visualize
secondary sprouts in isolation of primary ISVs. Indeed, in these
mutant embryos, aISVs fail to form, while secondary sprouts
emanate normally.16 Synectin silencing in PLC␥1y10xFli1:
eGFPy1 embryos showed that only half the normal number of
secondary sprouts developed by 48 hpf (sprouts in 10 somites:
7.3 ⫾ 0.9 in control vs 4.0 ⫾ 0.8 in synectinKD; N ⫽ 53-57
embryos, P ⬍ .01; Figure 4F-H). Thus, also with this method,
approximately 50% fewer secondary sprouts branched off in
synectinKD embryos.
Silencing of synectin does not alter secondary
angiogenic sprouting
We then assessed whether synectin silencing impaired lymphangiogenic sprouting using an indirect method (ie, by counting the
number of ISVs that were connected to the PCV [vISVs]), as the
latter are established when angiogenic secondary sprouts connect
to primary ISVs. Because a comparable fraction of lymphangiogenic and angiogenic sprouts normally emanates from the PCV, a
normal number of vIVSs, in combination with a reduced total
number of secondary sprouts, would be evidence for defective
formation of lymphangiogenic sprouts. In control 4 dpf Fli1:
eGFPy1 embryos (N ⫽ 29), 55% of the ISVs were connected to the
PCV and thus venous, while the remaining fraction was connected
to the DA and hence arterial (Figure 4I). Notably, in the most
severely affected synectinKD embryos, lacking the PL completely
or forming only a partial PL in 30% of the somites (N ⫽ 18), half of
the ISVs was still connected to the PCV (Figure 4I), showing that
angiogenic sprouts normally emanated from the PCV and implying
that lymphangiogenic sprouting was impaired in synectinKD embryos. As a lymphangiogenic sprouting defect might reflect a defect
in migration and/or emergence of the lymphatic lineage, but the
dynamics of Prox-1 expression during lymphatic differentiation
can be better visualized in frog than zebrafish embryos, we resorted
to tadpoles, a previously validated model to study lymphangiogenesis,17 to dissect the underlying mechanism in more detail.
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3360
HERMANS et al
Figure 3. Knockdown of synectin disrupts thoracic duct formation in zebrafish.
In all panels, the head of the embryo faces left and dorsal is up. Scale bars represent
100 ␮m in panels A-B and 50 ␮m in panels C-D. DA indicates dorsal aorta; and PCV,
posterior cardinal vein. (A-D) Confocal images of GFP⫹ vessels in the trunk of 7-dpf
Fli1:eGFPy1 zebrafish embryos, showing the formation of a normal lymphatic thoracic duct
(TD) in the control embryo (A,C) but not in the synectinKD embryo (B,D). Panels C and
D represent close-ups of the boxed areas in panels A and B, between the dorsal aorta
(DA) and posterior cardinal vein (PCV). In these latter panels, arrows highlight the TD, while
asterisks denote absence of TD. (E) Quantification of the TD formation defects after
injection of SynATG1 at 7 dpf. Percentages of embryos displaying complete lack of TD,
TD formation over 10%-30% or 30%-90% of its normal length, and a normal TD are
represented for each treatment group (see also supplemental Table 3). We quantitatively
analyzed TD formation by scoring its presence in 10 consecutive somite segments (from
somite 5 to somite 15).
Knockdown of synectin impairs lymphangiogenesis in tadpoles
We first analyzed synectin expression in tadpoles. In agreement
with previous findings,28 whole-mount in situ hybridization at stage
35-40 revealed expression of synectin in the neural tube, eye, brain,
otic vesicle, somites, and branchial arches, as well as at locations of
the DA, PCV, and dorsal longitudinal anastomosing vessel (DLAV),
where the ventral caudal lymph vessel (VCLV) and dorsal caudal
lymph vessel (DCLV) develop in the frog embryo (not shown and
supplemental Figure 5A-F). We also used a novel ‘LEC labeling’
technique in transgenic Flk1:eGFP frogs, that express eGFP in
blood and lymph vessels (generation of this line will be reported
elsewhere). Upon intracardial injection and extravasation from
blood vessels, tetramethyl rhodamine isothiocyanate (TRITC)dextran is selectively taken up by LECs, allowing isolation of
doubly labeled yellow LECs and singly labeled green BECs by
flow cytometry. RT-PCR showed that sorted BECs and LECs
expressed synectin (synectin/105 ␤-actin mRNA copies: 874 ⫾ 625
for BECs vs 1415 ⫾ 822 for LECs; N ⫽ 4).
We then evaluated whether silencing of synectin impaired
lymphatic development. In tadpoles, BECs from the PCV, express-
BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
ing Prox-1, migrate ventrally to form the VCLV, or dorsally to the
level of the DLAV to establish the DCLV.17 To knockdown
synectin, Flk1:eGFP tadpoles were injected with the nonoverlapping morpholinos xSynATG1 or xSynATG2, targeting the ATG of the
Xenopus laevis synectin mRNA. As both morpholinos blocked
translation (supplemental Figure 6) and caused indistinguishable
phenotypes (supplemental Table 5), only results for xSynATG1 are
shown. We first determined a range of submaximal morpholino
doses, that minimally affected vascular development. Screening of
GFP⫹ tadpoles at stage 45 (5 dpf) showed that, at 30 ng morpholino
per embryo, 17% of GFP⫹ tadpoles had only subtle vascular
defects (reduced complexity of capillary network interconnecting
ISVs; Figure 5A-B and supplemental Table 5). In line herewith,
blood flow in axial vessels was normal in most tadpoles (supplemental Table 5).
To characterize lymphatic development, we focused on the
formation of the VCLV and DCLV. LECs in Flk1:eGFP tadpoles
were labeled with TRITC-dextran to distinguish them from GFP⫹
BECs. Imaging of control tadpoles at stage 47 revealed that the
DCLV and VCLV formed a regular continuous vessel over its entire
length (Figure 5A,C,E). By contrast, in 57% of synectinKD tadpoles, the DCLV was incompletely formed and dysmorphogenic,
consisting of isolated LEC clusters, distributed discontinuously
over the trunk (Figure 5B,D and supplemental Table 5). The VCLV
appeared largely normal in most synectinKD tadpoles, at least
morphologically, while other morphants showed an irregular
structure (Figure 5B,F). Lymphangiography at stage 45 revealed
that the DCLV and VCLV were functional in all control tadpoles
(not shown and Figure 5G). When analyzing the DCLV in
15 synectinKD tadpoles, the dye was not drained at all or only over a
short distance, indicating that this lymphatic was dysfunctional
(not shown). Similarly, despite its apparent normal morphologic
appearance, the VCLV failed drain the dye (Figure 5H). We
previously noticed that the DCLV is more severely affected than
the VCLV upon silencing of Vegfr3,17,31 Vegfc (unpublished) or
Liprin␤-1,32 presumably because LECs, arising from the PCV, have
to migrate further to form the distant DCLV than the nearby VCLV.
In line with these findings, 32% of synectinKD tadpoles suffered
lymphedema around the heart and gut (Figure 5I-J and supplemental Table 5). Thus, synectin also regulates lymphatic development
in the tadpole model.
Synectin regulates lymphatic differentiation and migration
in tadpoles
The tadpole model allows study of the effects on LEC differentiation and migration.17 To explore whether lymphatic differentiation
or migration were impaired in synectinKD tadpoles, we stained
stage 35/36 embryos by whole-mount in situ hybridization for
Prox-1 to measure the accumulation of Prox-1⫹ cells in the ventral
trunk as an index of lymphatic lineage emergence, and in the more
dorsal trunk area as a measure of LEC migration using previously
established methods.17 Morphometric quantification revealed a
dose-dependent decrease of both Prox-1⫹ areas in synectinKD
tadpoles (by 40% at the highest morpholino dose; Figure 5K-N).
Thus, synectin regulates both lymphatic lineage formation and
LEC migration in tadpoles.
Synectin genetically interacts with Vegfc/Vegfr3
We then explored how synectin regulated LEC migration, using the
Fli1:eGFPy1 zebrafish model. Among the possible interacting
partners of synectin, we focused on neuropilin-2 (Nrp2), as it
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BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
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Figure 4. Knockdown of synectin impairs early lymphatic development in zebrafish. In all panels, the head of the embryo faces left and dorsal is up. Scale bars represent
50 ␮m. DA indicates dorsal aorta; ISV, intersomitic vessel; and PCV, posterior cardinal vein. (A-B) Confocal images of 60-hpf control (A) and synectinKD (B) Fli1:eGFPy1
embryos revealing a reduced formation of the parachordal lymphangioblast (PL) string (arrows in A) upon synectin knockdown. Asterisks in panel B denote absence of PL cells.
(C) Quantification of the PL cells in control and synectinKD Fli1:eGFPy1 zebrafish embryos at 60 hpf. The percentages of embryos lacking all PL cells and displaying PL string
formation over 10%-30%, 30%-90%, and 100% of its normal length are indicated per treatment group (see also supplemental Table 4). Formation of the PL was scored per
somite in 10 consecutive somites between somites 5 and 15. (D-E) Whole-mount in situ hybridization of 48-hpf control (D) and synectinKD (E) embryos for Tie-2, labeling all
secondary sprouts (arrows) emerging from the PCV. In synectinKD embryos the number of secondary sprouts was markedly reduced, by approximately 50%. Panels D⬘ and
E⬘ are magnifications of panel D and E, respectively. In synectinKD embryos somites lacking a secondary sprout are indicated with an asterisk. (F-G) Confocal images of 48-hpf
Fli1:eGFPy1xPLC␥1y10 control (F) and synectinKD(G) embryos showing a reduction of secondary sprouts upon synectin knockdown. Small arrows indicate unilateral secondary
sprouts; long arrow denotes PL cells. (H) Quantification of the number of unilateral secondary sprouts in a 10-somite region of 48-hpf Fli1:eGFPy1xPLC␥1y10 embryos
confirmed a significant reduction of nearly 50% in secondary sprouts budding from the PCV upon synectin knockdown. (I) Quantification of the fraction of venous ISVs,
identified by their connection to the PCV upon confocal screening, in a 10-somite region in 4-dpf Fli1:eGFPy1 embryos revealed a normal ratio of venous ISVs upon synectin
knockdown. Error bars in panels H-I represent SEM; *P ⬍ .05 by univariate analysis.
contains a PDZ-domain33 and regulates, as a coreceptor of
Vegfr3,34,35 lymphatic sprouting in mice.36,37 Vegfc stimulates LEC
migration through Vegfr3 signaling in mice,1,38,39 zebrafish20,40 and
tadpoles.17,31 Because Vegfr3 is the signaling receptor of the
Vegfr3/Nrp2 complex, and silencing of synectin phenocopies the
lymphatic defects induced by silencing of Vegfc or Vegfr3 in
zebrafish20,40 and tadpoles,17,31 we first assessed whether synectin
and Vegfr3 genetically interacted, by testing whether a combination
of low morpholino doses of synectin (2.5 ng/embryo) and Vegfr3
(1.25 ng/embryo), which by themselves induced only a minimal
effect, caused a more significant lymphatic defect (the low
morpholino doses were denoted by L-KD, as in synectinL-KD, to
distinguish them from the dose used in the incomplete silencing
studies). To silence Vegfr3, we used the previously characterized
Vegfr3ATG1 morpholino.40 This analysis revealed that, compared
with each single knockdown, the combined knockdown caused more
severe defects in TD and PL formation in synectinL-KDxVegfr3L-KD
morphants (P ⬍ .05 vs single knockdown; Figure 6A-B).
We also assessed whether synectin genetically interacted with
Vegfr3 in regulating lymphangiogenic sprouting in the PLC␥y10
zebrafish model. A low SynATG1 dose did not reduce the number of
secondary sprouts (7.4 ⫾ 0.3 in control vs 7.7 ⫾ 0.3 in SynL-KD;
N ⫽ 49-62, P ⫽ .40; Figure 6C), while a low dose of Vegfr3ATG1
slightly inhibited secondary sprouting (5.9 ⫾ 0.3 in Vegfr3L-KD;
N ⫽ 49-62, P ⬍ .001; Figure 6C). However, the combination of
both morpholinos impaired secondary sprouting significantly more
(4.9 ⫾ 0.2 in synectinL-KDxVegfr3L-KD; N ⫽ 70, P ⬍ .001 vs synectinL-KD; P ⬍ .05 vs Vegfr3L-KD; Figure 6C). We therefore assessed
whether angiogenic or lymphangiogenic secondary sprouts were
affected in synectinL-KDxVegfr3L-KD embryos, by counting the
number of vISVs in 4 dpf Fli1:eGFPy1 embryos (for rationale, see
above).
In line with reports that Vegfr3 regulates angiogenic secondary
sprouting,40 the fraction of vISVs was reduced by a low dose of
Vegfr3ATG1 but not of SynATG1 (% vISVs: 26 ⫾ 12% in Vegfr3L-KD
vs 52 ⫾ 7% in synectinL-KD or 54 ⫾ 9% in control; N ⫽ 17-29;
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HERMANS et al
BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
Figure 5. Synectin knockdown impairs lymphatics in tadpoles. In all panels, the head of the tadpoles faces left and dorsal is up. Scale bars represent 100 ␮m in panels
A-B and K-L; 50 ␮m in panels C-F; 150 ␮m in panels G-H; and 400 ␮m in panels I-J. DCLV indicates dorsal caudal lymph vessel; DLAV, dorsal anastomosing longitudinal
vessel; PCV, posterior cardinal vein; and VCLV, ventral caudal lymph vessel. (A-F) Fluorescent analysis of blood and lymph vessels in stage 47 transgenic Flk1:eGFP tadpoles.
Lymph vessels are labeled with TRITC-dextran that was intracardially injected at stage 45 and taken up by LECs after extravasation from the blood vessels. Hence, GFP⫹
BECs can be easily distinguished from doubly labeled (yellow) LECs. Areas in the trunk corresponding to the boxed areas in panels A or B are shown in panels C-F. In control
tadpoles, both the DCLV (C) and the VCLV (E) developed normally. Injection of 30 ng of synectin morpholino resulted in a hypoplastic, disorganized, and discontinuous DCLV
(D), while the VCLV (F) had a grossly normal appearance. (G-H) Lymphangiography of stage 45 Flk1:eGFP tadpoles, revealing dysfunction of the VCLV after synectin
knockdown (H) in contrast to normal controls (G). Whereas in control injected embryos, the dye is drained normally in a rostral direction (white arrows in G), no dye uptake was
observed in the VCLV in synectinKD tadpoles. White asterisks indicate injection site of the fluorescent dye. (I-J) Bright-field pictures of live embryos at stage 45 (5 dpf), showing
lymphedema (arrows) in a synectinKD tadpole (J) compared with a control tadpole (I). (K-L) Whole mount Prox-1 in situ hybridization of stage
35/36 tadpoles, revealing the presence of fewer Prox-1⫹ LECs in the area ventrally to the dorsal margin of the endoderm (reflecting lymphatic lineage emergence) and in the
area dorsally to this margin (reflecting LEC budding/migration) in the posterior trunk of synectinKD-morphant tadpoles (L) compared control (K) tadpoles. Dotted line: dorsal
margin of the endoderm. (M-N) Morphometric measurement revealed a dose-dependent decrease of the Prox-1⫹ area both ventrally (M) and dorsally (N) of the dorsal margin
of the endoderm in the trunk of Prox-1–stained synectinKD compared with control tadpoles at stage 35/36, indicating that lymphatic lineage development and migration is
affected upon synectin knockdown. Values expressed relative to control. *P ⬍ .001 by multivariate analysis.
P ⬍ .001). Notably, coknockdown of Vegfr3 and synectin did not
further reduce the fraction of vISVs (% vISVs: 25 ⫾ 12% in
synectinL-KDxVegfr3L-KD; N ⫽ 27; P ⫽ .968 vs Vegfr3L-KD), indicating that angiogenic sprouting was not under the control of a
synectin/Vegfr3 interaction. Thus, the normal number of angiogenic sprouts, in combination with the reduced total number of
secondary sprouts and impaired formation of PL and TD, in
synectinL-KDxVegfr3L-KD embryos indicates that the genetic synectin/
Vegfr3 interaction did not control angiogenic but lymphangiogenic
sprouting. Because other experiments revealed that Vegf/Vegfr2
signaling did not affect lymphatic development in zebrafish
(supplemental Note 2), we did not explore an interaction with
synectin.
Synectin genetically interacts with Nrp2a in vivo
We then analyzed whether synectin genetically interacted with
Nrp2. We first examined whether the zebrafish Nrp2a and Nrp2b
coorthologues regulated lymphatic development, using previously
validated morpholinos for Nrp2a (Nrp2aATG1) and Nrp2b
(Nrp2bATG1).41 Consistent with previous findings,42 a maximal dose
of 10 ng Nrp2aATG1 caused hind brain edema and arteriovenous
(AV) shunts in a small fraction of embryos, while a similar dose of
Nrp2bATG1 induced cardiac edema formation and mild DA dysmor-
phogenesis in some morphants (not shown). As reported,42 these
blood vessel phenotypes were confined to the extreme caudal
region of the trunk (not shown); morphant embryos with edema or
morphologic anomalies were excluded. The residual Nrp2aKD and
Nrp2bKD embryos developed normally by 6 dpf with no or only
minimal blood vessel or circulatory defects in the mid-trunk (where
we scored lymphatic development; ie, more rostral than the caudal
region in which AV shunts/DA defects were present; Figure 6E,G).
While lymphatic development was normal in Nrp2bKD embryos
(supplemental Figure 7A), Nrp2aKD dose-dependently caused PL
and TD defects (Figure 6D-K).
We then analyzed whether synectin genetically interacted with
Nrp2a. A low dose of Nrp2aATG1 (Nrp2aL-KD) or SynATG1 (SynL-KD)
minimally affected TD formation (Figure 6L). In contrast, knockdown of both genes by combined injection of a low dose of both
morpholinos severely impaired lymphatic development, as the TD
was absent in 30% of synectinL-KDxNrp2aL-KD morphants (P ⬍ .001
vs single knockdown; Figure 6L). Similar findings were obtained
when analyzing the PL (not shown).
Notably, however, Nrp2a knockdown did not affect secondary
sprout formation in PLC␥1y10xFli1:eGFPy1 embryos (supplemental Figure 7B). Coknockdown analysis did also not reveal a genetic
interaction between synectin and Nrp2a in secondary sprout
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BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
SYNECTIN IN LYMPHATIC DEVELOPMENT
3363
Figure 6. Synectin modulates Vegfr3 signaling in lymphatic development in vivo. In all panels, the head of the embryo faces left and dorsal is up. Scale bars represent
50 ␮m in panels D-E and 100 ␮m in panels F-I. DA indicates dorsal aorta; ISV, intersomitic vessel; and PCV, posterior cardinal vein. (A) Quantification of parachordal
lymphangioblast (PL) cell defects after injection of SynATG1 (2.5 ng; synL-KD; N ⫽ 79), Vegfr3ATG1 (1.25 ng; Vegfr3L-KD; N ⫽ 62) or both (N ⫽ 50) at 60 hpf. Percentages of
embryos displaying complete lack of PL cells, PL string over 10%-30% or 30%-90% of its normal length and a normal PL string are shown for each group. Compared with single
knockdown groups, the PL formation was more severely impaired in double morphants (P ⬍ .001). (B) Quantification of TD defects after injection of SynATG1 (2.5 ng; synL-KD;
N ⫽ 101), Vegfr3ATG1 (1.25 ng; Vegfr3L-KD; N ⫽ 107) or both (N ⫽ 86) at 7 dpf. Percentages of embryos displaying complete lack of TD, TD formation over 10%-30% or
30%-90% of its normal length and a normal TD are represented for each treatment group. Compared with single knockdown groups, the TD was more severely impaired in
double morphants (P ⬍ .001 vs synectinL-KD; P ⬍ .05 vs Vegfr3L-KD). (C) Quantification of the number of unilateral secondary sprouts in a 10-somite region of 48-hpf
Fli1:eGFPy1xPLC␥1y10 embryos revealed that coknockdown of synectin and Vegfr3 significantly aggravated the secondary sprouting defects compared with single knockdown
of either gene when using suboptimal doses of SynATG1 (2.5 ng; synL-KD) and Vegfr3ATG1 (2.5 ng; Vegfr3L-KD); (N ⫽ 45, 37, 48, and 63 for control, Vegfr3L-KD, synectinL-KD, and
coknockdown, respectively; *P ⬍ .05; **P ⬍ .01; ***P ⬍ .001). (D-E) Confocal images of 60-hpf control (D) and Nrp2aKD (E) Fli1:eGFPy1 embryos revealing impaired formation
of the PL string (arrows) upon Nrp2a knockdown. Asterisks denote absence of PL cells. (F-I) Confocal images of GFP⫹ vessels in the trunk of 7-dpf Fli1:eGFPy1 zebrafish
embryos, showing formation of a normal TD in a control embryo (F,H) but not in a Nrp2aKD embryo (G,I). Panels H and I represent close-up magnifications of the boxed areas in
panels F and G; arrows denote TD, asterisks denote absence of TD. (J) Quantification of PL cells in control and Nrp2aKD Fli1:eGFPy1 zebrafish embryos at 60 hpf. The
percentages of embryos lacking PL cells and displaying PL string over 10%-30%, 30%-90%, and 100% of its normal length are indicated per treatment group. Formation of the
PL string was scored per somite in 10 consecutive somites between somite 5 and 15 (N ⫽ 106, 152, and 60 for 0, 5, and 10 ng of Nrp2aATG1, respectively). (K) Quantification of
TD in control and Nrp2aKD Fli1:eGFPy1 zebrafish embryos at 7 dpf. The percentages of embryos lacking TD and displaying TD formation over 10%-30%, 30%-90%, and 100%
of its normal length are indicated per treatment group. Formation of the TD was scored per somite in 10 consecutive somites between somite 5 and 15 (N ⫽ 99, 164, and 56 for
0, 5, and 10 ng of Nrp2aATG1, respectively). (L) Quantification of TD formation after injection of Nrp2aATG1 (5 ng; Nrp2aL-KD; N ⫽ 74), SynATG1 (2.5 ng; synL-KD; N ⫽ 98) or both
(N ⫽ 41) revealed that coknockdown impaired lymphatic development more severely than single synectinL-KD (P ⬍ .001) or Nrp2aL-KD (P ⬍ .001).
formation (supplemental Figure 7E; see below for discussion).
Thus, our findings are consistent with a model, whereby synectin
genetically interacts with Vegfr3 and Nrp2a during lymphatic
development, but only beyond the initial stage of lymphangiogenic
sprouting.
Synectin mediates VEGFC/VEGFR3-driven LEC
sprouting in vitro
Because synectin regulates LEC migration in tadpoles and genetically interacts with Vegfr3, known to stimulate LEC migration,1,31,39 we examined whether synectin knockdown inhibited
sprouting of cultured HDLECs in response to vascular endothelial
growth factor C (VEGFC). We silenced synectin expression in
LECs using simultaneously 2 nonoverlapping synectin–specific
siRNAs. qRT-PCR analysis showed that synectin mRNA levels in
HDLECs were reduced by 80% (not shown). HDLECs treated with
control or synectin-specific siRNA were aggregated as spheroids
and lymphatic sprouts were visualized by CD31 immunostaining.
VEGFC stimulated lymphatic sprouting in control spheroids
(N ⫽ 17; Figure 7A,C,E), but synectinKD HDLECs formed substantially fewer sprouts in response to VEGFC (N ⫽ 26, P ⬍ .001;
Figure 7B,D-E).
Initial evidence for a role of synectin lymphatic differentiation
in vitro
The reduced number of Prox-1⫹ cells in the ventral region in
synectinKD tadpoles suggested a role for synectin in lymphatic
differentiation. In mice, Sox18 acts upstream of Prox-1 during this
process and lymphatic reprogramming is mimicked in vitro by
overexpression of Sox18 in venous ECs.3,6 We therefore overexpressed Sox18 in murine H5V BECs, transfected them with control
or synectin-specific siRNA at day 0, and analyzed lymphatic
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3364
HERMANS et al
BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
Figure 7. Synectin regulates Vegfc-driven lymphatic sprouting and Sox18-mediated lymphatic reprogramming in vitro. (A-D) CD31 immunostaining of untreated
(A-B) or VEGFC-stimulated (C-D; 200 ng/mL) LEC spheroids in fibrin gels, revealing reduced VEGFC sprouting response by LECs transfected with synectin-specific
(B,D; synectinKD) compared with control siRNA (A,C). Scale bar represents 100 ␮m. (E) Quantification of the sprout number per LEC spheroid, revealing that synectin
knockdown impaired lymphatic sprouting in response to VEGFC (N ⫽ 17 for control; N ⫽ 26 for synectinKD), while baseline sprouting of control (N ⫽ 17) and synectinKD LECs
(N ⫽ 35) was comparable. *P ⬍ .001 by univariate analysis. (F) RT-PCR of control and synectinKD H5V blood vascular endothelial cells (BECs), transfected with Sox18-virus
(BECsSox18) or GFP-virus (BECsGFP), revealing that knockdown of synectin impaired up-regulation of Prox-1 in BECsSox18 without affecting Nrp1 expression. Results are
represented as fold change vs control BECsGFP. Dashed line indicates baseline expression levels in BECsGFP. N ⫽ 3; *P ⬍ .01 by Student t test. Error bars represent SEM.
marker expression on day 7, as reported.6 The siRNA transfection
procedure was repeated on day 3 to maintain prolonged synectin
silencing until 7 days, when synectin mRNA levels were reduced
by 60% (not shown).
As reported,6 Prox-1 transcript levels were higher in BECs
transfected with Sox18-lentivirus (BECssox18) than control GFPvirus (BECsGFP), when treated with control siRNA; expression of
the blood vascular marker Nrp1 was not altered (Figure 7F). Upon
synectin knockdown, induction of Prox-1, but not of Nrp1,
expression in BECsSox18 was inhibited (Figure 7F). Knockdown of
synectin did not alter gene expression in BECsGFP (relative mRNA
levels vs control: 1.07 ⫾ 0.03 for Sox18; 0.84 ⫾ 0.12 for Prox1;
and 0.97 ⫾ 0.09 for Nrp1; N ⫽ 3; P ⬎ .05). Thus, silencing of
synectin impaired Sox18-driven lymphatic reprogramming in vitro.
Discussion
The present study provides genetic evidence for a role of synectin
in early lymphatic development. In zebrafish and frog embryos,
knockdown of synectin dose-dependently impaired and, in the
most severely affected embryos, even aborted lymphatic formation.
Several lines of evidence suggest that TD defects in synectinKD
zebrafish were due to defects in lymphangiogenic sprout formation
from the venous system. First, lymphangiogenic sprouts that give
rise to PL cells (ie, precursors of LECs forming the TD) were
underdeveloped or, in the most severely affected morphants, did
not form at all in synectinKD embryos. In the ccbe1, Vegfc, or Vegfr3
mutants, lack of formation of these lymphangiogenic sprouts also
aborts further lymphatic development.20,40 Second, the fraction of
synectinKD embryos without TD closely correlated with the fraction
of morphants without lymphangiogenic sprouts and PL cells. Third,
the missing secondary sprouts in synectinKD embryos were lymphangiogenic and not angiogenic, since these embryos developed
at the expected ratio vISVs, the formation of which requires normal
angiogenic secondary sprouting.
So far, silencing of only a few other genes (Ccbe1, Vegfc, or
Vegfr3) is known to abort lymphangiogenic sprouting, the formation of the PL and TD.20,40 However, in these morphant embryos,
branching of angiogenic sprouts from the PCV was also impaired.20
By contrast, in Dll4/Notch hypomorphants, the fraction of angiogenic sprouts is increased at the expense of lymphangiogenic
sprouts.11 It is thus noteworthy that synectin silencing selectively
impaired lymphangiogenic without affecting angiogenic secondary
sprouting. The identification of synectin as the first selective
regulator of lymphangiogenic sprouting suggests that formation of
lymphangiogenic versus angiogenic sprouts is under distinct genetic control.
Synectin silencing inhibited migration of cultured LECs in
response to VEGFC and reduced the dorsal Prox-1⫹ area, a
parameter of LEC migration, in tadpoles. Similar LEC migration
defects contribute to lymphatic impairment in tadpoles or mice
lacking VEGFC or VEGFR3.1,17,31,38,39 Moreover, silencing of
synectin in zebrafish aggravated the lymphatic defects, induced by
silencing of Vegfr3 as well as of Nrp2a, the coreceptor of
Vegfr3,34,35 that contains a PDZ-binding domain,33 through which
synectin interacts with its partners.27 These data thus illustrate a
genetic interaction of synectin with Vegfr3 as well as with Nrp2a.
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BLOOD, 28 OCTOBER 2010 䡠 VOLUME 116, NUMBER 17
While Vegfr3/synectin participated already in lymphangiogenic
sprouting, Nrp2a/synectin only regulated subsequent formation of
the PL and TD. In mice, Nrp2 is also dispensable for initial
lymphangiogenic budding from veins, but required for subsequent
lymphatic development.36 Together, these genetic studies suggest a
model whereby synectin modulates Vegfr3 signaling in lymphatic
development in a Nrp2a-independent manner (during lymphangiogenic sprouting) or Nrp2a-dependent manner (PL and TD
formation).
How synectin regulates Vegfr3 signaling is an intriguing
question. During arterial development, synectin promotes intracellular Vegfr2 trafficking to early endosomes, required for downstream signaling, via Nrp1-dependent and -independent mechanisms.13,43 By analogy with Vegfr2/Nrp1, it is tempting to speculate
that synectin might perhaps also participate in Vegfr3 endocytosis,
at least partially, via an interaction with Nrp2. Supporting this
model, Vegfr3 not only colocalizes with Nrp2 during endocytosis
upon stimulation by Vegfc and Vegfd,35 but internalization is also
required for proper Vegfr3 signaling.44 Because synectin modulates
Vegfr3-driven lymphangiogenic but not angiogenic sprouting, it is
tempting to speculate that synectin-mediated endocytosis regulates
distinct Vegfr3 signaling pathways. Elucidation of the underlying
molecular details will require future study.
Lymphatic defects were unlikely to be secondary to angiogenic
alterations, as the PCV formed normally and expressed proper
venous markers in synectinKD embryos, consistent with findings
that synectin is dispensable for venous morphogenesis.12 In addition, the DA and primary aISVs formed normally and expressed
arterial markers upon incomplete silencing of synectin. In fact,
alterations in arterial identity or morphogenesis are unlikely to
perturb lymphangiogenic sprouting or cause the type of lymphatic
defects, observed upon synectin knockdown, since secondary
sprouts and PL cells formed normally in PLC␥1y10 mutants, which
even lack primary ISVs and exhibit an underdeveloped and
improperly differentiated DA.16,25 In addition, we and others22
observed that lymphatic development is nearly normal upon
silencing of Vegfaa- or Kdr-like, 2 well-established regulators of
arterial morphogenesis and specification.45,46
A limitation of our study is that Prox-1 levels in the PCV at the
time of lymphatic differentiation in zebrafish are below the
threshold of detection (unpublished findings, P.C. and S.S.M.;
personal communication, H. Gerhardt [London Research Institute,
Cancer Research UK, London, United Kingdom] and F. Cotelli
[University of Milan, Milan, Italy]), thereby precluding us from
analyzing whether synectin silencing impairs LEC differentiation
in zebrafish. We therefore used alternative models in vivo and in
vitro. Indeed, our genetic analysis in tadpoles shows that silencing
of synectin caused an underdevelopment of Prox-1⫹ cells at sites
SYNECTIN IN LYMPHATIC DEVELOPMENT
3365
previously implicated in differentiation of LECs from venous
ECs.17 In addition, silencing of synectin reduced lymphatic reprogramming of venous ECs in vitro upon overexpression of Sox18,
an established regulator of lymphatic differentiation in mice.6 The
role of Sox18 in lymphatic development in tadpoles and zebrafish
has not yet been documented but, interestingly, silencing of Sox18
(in combination with Sox7) in zebrafish impaired venous ECs from
which LECs differentiate.47-49 Notably, because PDZ-dependent
binding has been reported for some of the SoxA family members,50
a possible interaction of synectin with Sox18 is an exciting
possibility that remains to be further investigated.
In conclusion, synectin regulates lymphangiogenesis in zebrafish and tadpoles through genetic interactions with Vegfr3 and
Nrp2a.
Acknowledgments
The authors thank K. Brepoels, A. Carton, M. De Mol, E. Janssens,
S. Louwette, A. Manderveld, M. Peeters, J. Souffreau, B. Tembuyser,
A. Van den Eynde, A. Van Nuffelen, B. Vanwetswinkel,
S. Verstraeten, S. Vinckier, and S. Wyns for technical assistance.
K.H., W.V., K.A., I.G., and F.D.S. are sponsored by a PhD grant
of the Institute for the promotion of Innovation through Science
and Technology (IWT) in Flanders (IWT-Vlaanderen), Belgium.
E.G. and S.S.-M. are supported by the Royal Netherlands Academy
of Arts and Sciences (KNAW). This work is supported by
long-term structural Methusalem Funding by the Flemish Government to P.C., Research Foundation Flanders (FWO) Research
Project Funding by the Flemish Government to P.C., Interuniversity Attraction Poles–Belgian Science Policy (IUAP P6/20) to
M.D., and European Commission Lymphangiogenomics Consortium Funding (LSHG-CT-2004-503573) to P.C.
Authorship
Contribution: K.H., F.C., A.N., M.A., M.S., M.D., S.S.-M., E.D.,
K.A., and P.C. designed research; K.H., F.C., W.V., W.Z., I.G., F.O.,
A.N., F.D.S., E.G., K.A., and B.R. performed the experiments and
analyzed the data; D.K. provided vital reagents; and K.H., F.C.,
M.D., and P.C. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Peter Carmeliet, Vesalius Research Center,
Vlaams Iustituut voor Biotechnologie, Katholieke Universiteit
Leuven, Campus Gasthuisberg, Herestraat 49, B-3000, Belgium;
e-mail: [email protected].
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2010 116: 3356-3366
doi:10.1182/blood-2009-11-254557 originally published
online July 14, 2010
Role of synectin in lymphatic development in zebrafish and frogs
Karlien Hermans, Filip Claes, Wouter Vandevelde, Wei Zheng, Ilse Geudens, Fabrizio Orsenigo,
Frederik De Smet, Evisa Gjini, Kristof Anthonis, Bin Ren, Dontcho Kerjaschki, Monica Autiero, Annelii
Ny, Michael Simons, Mieke Dewerchin, Stefan Schulte-Merker, Elisabetta Dejana, Kari Alitalo and
Peter Carmeliet
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