Mafba is a downstream transcriptional effector of Vegfc signaling essential for embryonic lymphangiogenesis in zebrafish

Abstract

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.
© 2015 Koltowska et al.; Published by Cold Spring Harbor Laboratory Press.

Full text

mafba is a downstream transcriptional
effector of Vegfc signaling essential
for embryonic lymphangiogenesis
in zebrafish
Katarzyna Koltowska,
1
Scott Paterson,
1
Neil I. Bower,
1
Gregory J. Baillie,
1
Anne K. Lagendijk,
1
Jonathan W. Astin,
2
Huijun Chen,
1
Mathias Francois,
1
Philip S. Crosier,
2
Ryan J. Taft,
1
Cas Simons,
1
Kelly A. Smith,
1
and Benjamin M. Hogan
1
1
Division of Genomics of Development and Disease, Institute for Molecular Bioscience, The University of Queensland, St Lucia,
Brisbane, Queensland 4072, Australia;
2
Department of Molecular Medicine and Pathology, School of MedicalSciences, University
of Auckland, Auckland 1023, New Zealand
The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer
metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lym-
phatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lym-
phangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains
unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for
lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their
initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and
we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc
signaling increases mafba expression to control downstream transcription, and this regulatory relationship is de-
pendent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothe-
lium. Here we identify an indispensable Vegfc–SoxF–Mafba pathway in lymphatic development.
[Keywords: lymphatic; vascular; Mafb; Vegfc; Sox18; Sox7; zebrafish]
Supplemental material is available for this article.
Received April 2, 2015; revised version accepted July 1, 2015.
Lymphangiogenesis is the formation of new lymphatic
vessels from pre-existing vessels. Lymphangiogenesis
plays integral roles in cancer metastasis, lymphedema,
and cardiovascular disease (Lim et al. 2013; Martel et al.
2013; Stacker et al. 2014). Common molecular pathways
control lymphangiogenesis in development and disease,
and much of our current understanding has come from
the study of embryogenesis. In the embryo, the lymphatic
vasculature derives chiefly from pre-existing veins
through a process involving cellular transdifferentiation,
migration, and proliferation and vessel morphogenesis
(Oliver and Srinivasan 2010; Koltowska et al. 2013).
Lymphangiogenesis is dependent on VEGFR3 signaling
in all known contexts, including during development.
Knockout mice for Vegfr3 are embryonic-lethal due to car-
diovascular failure (Dumont et al. 1998), and heterozy-
gous mutation of Vegfr3 in the Chy mouse model leads
to lymphedema and lymphatic vascular defects (Karkkai-
nen et al. 2001). Furthermore, the transgenic overexpres-
sion of a soluble inhibitory (ligand trap) form of VEGFR3
disrupts tissue lymphangiogenesis (Makinen et al. 2001).
Mouse mutants for Vegfc fail to form the earliest lymphat-
ic sprouts from embryonic veins, and, indicative of the
instructive role for VEGFC, overexpression of this ligand
promotes ectopic tissue lymphangiogenesis (Jeltsch et
al. 1997; Karkkainen et al. 2004; Hagerling et al. 2013).
In humans, the VEGFC/VEGFR3 pathway also controls
lymphatic vessel development, and patients with muta-
tions in either VEGFR3 or VEGFC develop primary
lymphedema in familial Milroy’s disease or Milroy’s-
like lymphedema, respectively (Irrthum et al. 2000; Kark-
kainen et al. 2000; Gordon et al. 2013). Finally, several co-
receptors and modulators of VEGFC/VEGFR3 signaling
play crucial roles in lymphangiogenesis (for review, see
Corresponding author: b.hogan@imb.uq.edu.au
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.263210.
115.
© 2015 Koltowska et al. This article is distributed exclusively by Cold
Spring Harbor Laboratory Press for the first six months after the full-issue
publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml).
After six months, it is available under a Creative Commons License (At-
tribution-NonCommercial 4.0 International), as described at http://
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Schulte-Merker et al. 2011; Koltowska et al. 2013; Zheng
et al. 2014), underscoring how central the pathway is in
lymphatic development.
Characterization of the zebrafish lymphatic vasculature
has demonstrated the highly conserved function of the
VEGFC/VEGFR3 signaling pathway in vertebrates (Kuch-
ler et al. 2006; Yaniv et al. 2006). Forward genetic screens
for mutants lacking lymphatics in zebrafish discovered a
function for Ccbe1 (Hogan et al. 2009a), which was subse-
quently shown to play a conserved role in mice and hu-
mans (Alders et al. 2009; Connell et al. 2010; Bos et al.
2011). CCBE1 regulates the processing and activation of
immature VEGFC to its mature, functional form neces-
sary for lymphangiogenesis (Jeltsch et al. 2014; Le Guen
et al. 2014). In addition, zebrafish mutants have been de-
scribed in vegfc and vegfr3 themselves, and we now
know that zebrafish Vegfc signaling controls all secondary
angiogenesis, including the formation of lymphatic pre-
cursors (PLs) from the posterior cardinal vein (PCV) (Ho-
gan et al. 2009a,b; Villefranc et al. 2013; Le Guen et al.
2014). The coordinated activity of both Vegfc and Vegfd
further controls lymphangiogenesis of the facial lymphat-
ic network (Okuda et al. 2012; Astin et al. 2014).
Although the apical signaling pathway that governs
lymphatic sprouting is well established, we still have a
very limited understanding of how signaling controls
downstream pathways and regulates endothelial cell (EC)
transcription. Several transcription factors are known to
control the initial specification and maintenance of lym-
phatic EC (LEC) fate in mice, including PROX1, COUPT-
FII, and SOX18 (Wigle and Oliver 1999; Francois et al.
2008; Srinivasan et al. 2010; Srinivasan and Oliver 2011).
Other transcription factors controlling later differentia-
tion and specialization of lymphatic vessels include
FOXC2 and GATA2 (Petrova et al. 2004; Norrmen et al.
2009; Kazenwadel et al. 2012; Lim et al. 2012). Interesting-
ly, emerging data have suggested that both the mainte-
nance of PROX1 and the induction of SOX18 activity can
be driven by VEGF signaling mechanisms (Deng et al.
2013; Duong et al. 2014; Srinivasan et al. 2014). While
this integration of cell-extrinsic signaling and cell-intrin-
sic transcriptional information appears to play an impor-
tant role in lymphatic development, the spatiotemporal
control of signaling and the complexity of EC transcrip-
tional mechanisms remain far from fully understood.
The initial genetic screens performed for zebrafish lym-
phatic development used a pan-endothelial marker [Tg
(fli1a:EGFP)
y1
] and scored the presence of the thoracic
duct as a proxy for systemic lymphangiogenesis. We
took advantage of the recently described Tg(-5.2lyve1b:
dsRed) strain, which labels the comprehensive network
of developing lymphatic vessels and embryonic veins
(Okuda et al. 2012), to perform a phenotypically sensitized
forward genetic screen and integrated whole-genome se-
quence mapping for rapid gene discovery. Here, we report
the first mutant characterized from this screen, a mafba
mutant. We found that mafba is an essential Vegfc-regu-
lated transcription factor controlling lymphangiogenesis
and identified a role for SoxF transcription factors in in-
ducing mafba expression.
Results
uq
4bh
mutants fail to form lymphatic vasculature
We designed and performed an ENU mutagenesis screen
in zebrafish. Briefly, we mutagenized the Tg(-5.2lyve1b:
dsRed) strain and performed a classical F3 embryonic
screen in this background. We sequenced the genomes
of the transgenic mutagenized founders and our in-house
WIK strain, which we used to generate mapping crosses
with a predefined genomic variation. In total, we used
838 mutagenized F1 genomes (419 F2 families) with, on
average, four F2 in-crosses scored per family (or 573 ge-
nomes screened). We identified 34 mutants that gave lym-
phatic deficiency/venous sprouting defects at either of
two time points: 3 d post-fertilization (dpf) and 5 dpf.
The uq
4bh
mutant showed a highly selective reduction,
up to a complete loss, of lymphatic vessels in the embry-
onic trunk and face but formed a grossly normal blood vas-
culature (Fig. 1A; Supplemental Fig. 1B). Overall, the body
plan was normal except for an otic vesicle defect and ede-
ma by 7 dpf (Figs. 1B, 2D; Supplemental Fig. 1A). Quanti-
fication of the number of LECs in the trunk and face using
a nuclear marker of ECs coupled with Tg(-5.2lyve1b:
dsRed) demonstrated a significant reduction in mutants
(Fig. 1C–E). In the developing facial lymphatics, reduc-
tions were observed in medial facial lymphatics and bran-
chial arch lymphatics (Fig. 1D,E; Supplemental Fig. 1C).
The number of LECs in the otolithic lymphatic vessel
was increased but was presumed to reflect altered, local
tissue patterning. Earlier in development, examination
of parachordal PL cell number showed no difference in
mutants (Fig. 1F,G). We analyzed the expression of prox1a
in the Tg(prox1a:Kalt4-4xUAS:uncTagRFP) transgenic
line that has been previously reported (Dunworth et al.
2014; van Impel et al. 2014) crossed onto a Tg(10xUAS:Ve-
nus) reporter strain. Interestingly,we saw no change in
prox1a expression in sprouts leaving the PCV, suggesting
normal establishment of cell identity in mutants (Fig. 1H,
I). Overall, these observations indicate that the uq
4bh
mu-
tants initiate lymphatic fate and sprouting secondary an-
giogenesis, but ongoing formation of a comprehensive
lymphatic network fails.
uq
4bh
is a mafba mutant
We used a whole-genome sequence-based approach to
map the genomic location of the affected gene (Supple-
mental Material; data not shown). We identified a region
of homozygosity on chromosome 23, and, within this
linked region, a candidate mutation was identified in the
mafba gene (Fig. 2A; Supplemental Fig. 2A). A C/T non-
sense mutation was identified (encoding Q155∗) and pre-
dicted to truncate Mafba prior to the critical basic region
leucine zipper (BRLZ) domain and thus is a predicted
loss-of-function allele (Fig. 2B,C). The zebrafish neural
segmentation mutant valentino (Moens et al. 1996) is a
mafba mutant and displays the same otic vesicle pheno-
type as uq
4bh
(Fig. 2D), suggesting a causative mafba mu-
tation. MAFB is a bZIP transcription factor that can act
as an activator or repressor. MAFB homologs regulate
Mafb in lymphangiogenesis
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cellular differentiation in various developmental con-
texts, controlling posterior hindbrain/otic fate decisions
(Moens et al. 1996; Moens and Prince 2002), podocyte
development (Sadl et al. 2002; Moriguchi et al. 2006), pan-
creatic β-cell differentiation (Artner et al. 2007), and he-
matopoietic lineage decisions (Sieweke et al. 1996, 1997;
Kelly et al. 2000; Bakri et al. 2005). However, MafB has
no previously described role in either lymphangiogenesis
or vascular development.
To confirm that the mutation in mafba causes the
lymphatic vascular developmental phenotype, we used
CRISPR genome editing to generate germline mosaic
zebrafish transmitting mutations in mafba (Supplemental
Fig. 2B). Mosaic founders were then crossed to carriers
for the uq
4bh
mutation, and the progeny were analyzed
for phenotypes. Compound heterozygous embryos (con-
firmed by genotyping) displayed the described valentino
otic phenotype, and this was coincident with a loss of
lymphatic vascular development (Fig. 2D,E). This comple-
mentation test confirmed the mutant as mafba
uq4bh
(Fig.
2E; Supplemental Fig. 2C,D).
mafba is expressed in the PCV and enriched
in secondary sprouts
To determine the cell type in which mafba is active, we
examined gene expression by in situ hybridization (ISH).
mafba expression was observed in neurons, rhombo-
meres, and the pancreas (data not shown) as well as the
PCV. We observed weak PCV expression at 24 h post-fer-
tilization (hpf), which was increased by 30 and 36 hpf, pre-
ceding and concomitant with secondary angiogenesis (Fig.
3A; Supplemental Fig. 3A). At 48 hpf, when secondary
sprouts have formed, we observed enriched expression of
mafba dorsally in ECs sprouting or sprouted from the
PCV (Fig. 3A). As ISH is insensitive at later developmental
stages, we examined mafba expression in FACS (fluores-
cent-activated cell sorting)-sorted populations of arterial
ECs (AECs), venous ECs (VECs), and LECs that we isolat-
ed from 60 hpf, 3 dpf, and 5 dpf embryos (Fig. 3B,C; Coxam
et al. 2014, 2015). Indicative of lineage-restricted expres-
sion in the vasculature, we found that mafba was highly
enriched in VECs compared with AECs at early time
Figure 1. The uq
4bh
mutant fails to form a lymphat-
ic vasculature. (A) Vascular nuclei [Tg(fli1a:
nlsEGFP); green] and veins and lymphatic vessels
[Tg(lyve1:DsRed); white] in the trunk of sibling (top
panels) and mutant (bottom panels) embryos at
5 dpf. (TD) Thoracic duct; (DLLV) dorsal longitudinal
lymphatic vessel; (ISLV) intersegmental lymphatic
vessel. Arrows indicate lymphatics, and asterisks in-
dicate their absence. Bars, 50 μm. (B) Gross morphol-
ogy of sibling (top) and mutant (bottom) embryos at
5 dpf. (C) Quantification of total LEC number using
the overlay of transgenic marker expression from A.
Mean ± SEM; scored siblings, n= 15; uq
4bh
,n= 18; t-
test. (∗∗∗∗)P< 0.0001. (D) The facial lymphatic net-
work in sibling (top) and mutant (bottom) embryos
at 5 dpf. (LFL) Lateral facial lymphatic; (OLV) otolithic
lymphatic; (LAA) branchial arch lymphatics; (MFL)
medial facial lymphatics. Bars, 50 μm. (E) Quantifica-
tion of facial lymphatic cell numbers in individual
embryos using the overlay of transgenic marker ex-
pression from A. Mean ± SEM; scored siblings, n= 6;
uq
4bh
,n= 8; t-test. (∗)P< 0.05. (F) PLs in the horizon-
tal myoseptum in sibling (top) and mutant (bottom)
embryos. Bars, 30 μm. (G) Quantification of PL num-
bers in individual embryos using the overlay of trans-
genic marker expression from A. Mean ± SEM; scored
siblings, n= 10; uq
4bh
,n= 12; t-test. (ns) No sig-
nificant difference. (H)prox1a-expressing LECs as
they emerge from the PCV at 36 h post-fertilization
(hpf) in sibling (top) and mutant (bottom) embryos
labeled by Tg(prox1a:Kalt4-4xUAS:uncTagRFP);Tg
(10xUAS:Venus). (Green) α-GFP. Bars, 30 μm. (I)
Quantification of prox1a-expressing LECs in individ-
ual embryos at 36 hpf. Mean ± SEM; scored siblings,
n= 16; uq
4bh
,n= 15; t-test. (ns) No significant
difference.
Koltowska et al.
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points (comparable with known VEC markers) and was
enriched in LECs compared with VECs by 5 dpf (compara-
ble with known venous and lymphatic markers lyve1b
and prox1a). We examined expression of other Maf family
genes and the duplicate Mafb homolog mafbb, which
showed detectable EC expression, suggesting that it could
play a compensatory role (Supplemental Fig. 4B,C); how-
ever, given the phenotype of mafba mutants, any compen-
sation must be partial.
mafba acts cell-autonomously during zebrafish
lymphangiogenesis
mafba expression is suggestive of a cell-autonomous func-
tion; hence, we performed cellular transplantation exper-
iments to formally test autonomy. We transplanted wild-
type Tg(fli1a:EGFP) cells into Tg(-5.2lyve1b:DsRed) re-
cipients from a mafba heterozygous in-cross and scored
for the presence of grafted (EGFP-positive) cells in arteries
(AECs), veins (VECs), and lymphatics (LECs) at 5 dpf. We
found that wild-type cells could as readily contribute
AECs, VECs, and LECs in mutant embryos (n= 13 vascu-
lar grafts) as they could in sibling controls (n= 11 vascular
grafts) (Fig. 4A,B). We traced all grafted cells with dextran
and noted the positions of non-ECs, which were in vari-
able locations and not consistent in rescued mutant em-
bryos. Together, these analyses suggest that mafba is
sufficient in ECs to direct lymphangiogenesis in mutant
embryos.
Reciprocally, we transplanted mutant cells from Tg
(kdrl:EGFP);Tg(-5.2lyve1b:DsRed) double-transgenic em-
bryos into unlabeled wild-type hosts. In this experimental
setting, AECs will express only EGFP, VECs will express
EGFP plus dsRED, and LECs will express only dsRED in
successful vascular grafts. Transplanted mutant cells
contributed to AECs (n= 12/12 vascular grafts) and VECs
(n= 3/12 vascular grafts) but not Tg(lyve1:DsRed)-ex-
pressing LECs (Fig. 4C,D). Interestingly, in one trans-
planted embryo (n= 1/12), mutant cells formed a section
of vasculature that appeared to be lymphatic based on
Figure 2. Positional cloning of mafba
uq4bh
using whole-genome sequence-based ho-
mozygosity mapping and mutation detec-
tion. (A) Schematic plot of genomic
homozygosity across all 25 chromosomes
(top), chromosome 23 (middle), and the re-
gion of linkage (bottom) (see the Materials
and Methods for details). The mafba gene
is located centrally within the region of
highest homozygosity. (B) Sequence chro-
matograms confirming the nonsense allele
in the mafba gene. (Top) Wild type (n=6
reads). (Middle) Heterozygous (n= 18
reads). (Bottom) Mutant (n= 19 reads). (C)
Schematic of the location of the Q155∗al-
lele predicted to truncate the critical
BRLZ domain of Mafba. (D) Otic vesicle
morphology of sibling (left) and mutant
(right) embryos at 3 dpf. Bars, 80 μm. (E)
Confirmation of causative mafba
uq4bh
al-
lele by complementation test with a
mafba
5 bp del
transmitting founder. Vascu-
lature visualized by Tg(kdrl:EGFP) (green)
and Tg(-5.2lyve1b:DsRed) (red) in the sib-
ling (top) and transheterozygous maf-
ba
uq4bh
;mafba
5 bp del
(bottom). Siblings, n
= 10; mafba
uq4bh
; mafba
5bpdel
,n= 10.
(Right) Red channel. Bars, 30 μm. See also
Supplemental Figure 2.
Mafb in lymphangiogenesis
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morphology, but this vessel expressed Tg(kdrl:EGFP),
which is normally restricted to blood vessels. Hence,
this grafted vessel was considered to have a differentia-
tion defect (Supplemental Fig. 3B). Taken together, re-
ciprocal cellular mosaic experiments demonstrate that
mafba acts autonomously in ECs during lymphatic vessel
development.
A set of mafba-dependent endothelial genes
are responsive to Vegfc signaling
We next isolated Tg(kdrl:EGFP)-expressing ECs from sib-
ling and mafba
uq4bh
mutant embryos at 48 hpf by embryo
dissociation and FACS for EGFP (Supplemental Fig. 4A).
We performed RNA sequencing (RNA-seq) in triplicate
and generated a concordant data set identifying 23
down-regulated and 68 up-regulated genes (Fig. 5A; Sup-
plemental Table 1). These dysregulated genes were gener-
ally lowly expressed, probably indicative of low numbers
of cells derived from secondary sprouts (which express
mafba) in the larger pool of Tg(kdrl:EGFP)-expressing
ECs. To confirm that these genes were dysregulated, we
used quantitative PCR (qPCR) for 15 down-regulated
and 47 up-regulated genes and confirmed that the majori-
ty was misexpressed, as indicated in the RNA-seq data
(Fig. 5B).
To understand how selective to secondary angiogenesis
these genes are, we sorted ECs using FACS from 30-hpf
MO-vegfc knockdown embryos (prior to venous sprout-
ing) and 48-hpf Tg(prox1a:Kalt4-4xUAS:uncTagRFP);Tg
(10xUAS:Vegfc) “vegfc-induced”embryos, which over-
express Vegfc in all prox1a-expressing tissues and hence
show ectopic venous angiogenesis (Helker et al. 2013;
Le Guen et al. 2014; K Koltowska, AK Lagendijk, C
Pichol-Thievend, JC Fischer, M Francois, EA Ober, AS
Yap, and BM Hogan, in prep.). We then performed qPCR
for 15 genes that were down-regulated in mafba mu-
tants. Of these 15 genes, 13 were down-regulated in
MO-vegfc ECs, and eight were up-regulated in Vegfc-in-
duced ECs (Fig. 5C). This observation suggested that, in
ECs, mafba-dependent genes are also dependent on nor-
mal Vegfc signaling.
mafba expression is up-regulated by Vegfc
We investigated mafba expression in MO-vegfc and
MO-vegfr3 embryos at 30 hpf and saw no change in mafba
expression (Supplemental Fig. 4D). At 48 hpf, when mafba
normally becomes enriched in the dorsal PCV, this en-
richment was lost in MO-vegfc and MO-vegfr3 embryos,
and overall EC mafba levels (normalized) were reduced
by qPCR (Fig. 5D,E). Venous sprouts fail to form in MO-
vegfc and MO-vegfr3 embryos, and so the loss of ex-
pression may be a consequence of failed morphogenesis.
However, the observation is consistent with selective
enrichment in cells responding to Vegfc signaling, so we
next examined mafba expression in vegfc-induced embry-
os. We observed vastly increased mafba expression in the
vasculature of vegfc-induced embryos by ISH and con-
firmed this increase by qPCR (normalized) using FACS-
sorted ECs from vegfc-induced embryos (Fig. 5F,G). We
did not find any evidence for regulation of vegfc,vegfr3,
or other Vegf family downstream from mafba (Supple-
mental Fig. 4E–G).
mafba regulates LEC migration from the horizontal
myoseptum but is dispensable for vegfc-induced
proliferation
To determine the earliest cellular defect in trunk lym-
phangiogenesis in mafba mutants, we time-lapse-imaged
PLs in the horizontal myoseptum from 48 hpf onward
in Tg(-5.2lyve1:DsRed);Tg(kdrl:EGFP) double-transgenic
embryos. While wild-type PLs actively migrated out of
the myoseptum and elongated along their arterial sub-
strates (Bussmann et al. 2010; Cha et al. 2012), mutant
PLs commonly failed to migrate from the myoseptum
(Fig. 6A; Supplemental Movies 1, 2). Some PLs dis-
played distinctly broad and rounded morphology during
Figure 3. mafba is expressed in endothelium
during lymphangiogenesis. (A) Expression of
mafba in the zebrafish trunk at 24 hpf and 36
hpf (left) and 48 hpf (right). (Bottom right pan-
els) Higher-magnification images of secondary
sprouts (PCV; arrowheads). (Boxed area) High-
magnification region; (bracket) dorsal aorta
(DA). Bars, 100 μm. (B) Quantitative PCR
(qPCR) on FACS-sorted AEC versus VEC popu-
lations at 60 hpf; mafba expression is VEC-en-
riched, comparable with vegfr3 and in contrast
to AEC marker flt1. Mean ± SEM. Expression is
relative to kdrl.(C) qPCR on FACS-sorted LEC
and VEC populations from 3 dpf and 5 dpf; the
mafba expression profile is similar to LEC
markers prox1a and lyve1b. Mean ± SEM. Ex-
pression is relative to rpl13.
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migration, which was not observed in wild-type cells (Fig.
6A). Interestingly, we did not find any evidence for dysre-
gulation of chemokine signaling components that control
this migratory event (Cha et al. 2012) or ephrinb2a/ephB4,
which have been implicated in EC migration (Supplemen-
tal Fig. 5A–C). We next examined mafba mutants in the
vegfc-induced transgenic overexpression background and
found that vegfc overexpression induced the proliferation
of VECs at the same level in mafba mutants as in wild-
type transgenic embryos (Fig. 6B,C). Together, these data
show that mafba controls the ongoing migration of PLs af-
ter initial secondary angiogenesis but is not required for
vegfc transgene-induced venous proliferation.
Vegfc up-regulates mafba expression in a SoxF
transcription factor-dependent manner
We and others have previously shown that VEGF signal-
ing can control the activity of SOX18 in human ECs,
mice, and zebrafish (Deng et al. 2013; Duong et al.
2014). To determine whether SoxF family transcription
factors play a role in mafba induction, we examined
mafba expression in sox7/sox18 double morpholino
(dMO)-injected embryos. We found a strong reduction to
complete absence of mafba expression in the PCV at 30
and 48 hpf (Fig. 7A), an unexpected observation because
sox7/sox18 dMO embryos display expanded expression
of classical VEC markers (Herpers et al. 2008; Pendeville
et al. 2008). Given that SOXF transcription factors can re-
spond to VEGFs and that we observed that mafba expres-
sion is also Vegfc-responsive, we next examined whether
the up-regulation of mafba by Vegfc is dependent on
SoxF transcription factors. We found that knockdown of
sox7 alone did not have an impact on the induction of
mafba by Vegfc (Fig. 7B,C). sox18 knockdown strongly
reduced the intensity of induction, and the dMO knock-
down further reduced the induction of mafba expres-
sion (Fig. 7B,C). Supporting this regulatory relationship
in vitro, transfection of human umbilical vein ECs with
SOX18 but not SOX7 induced MAFB expression (Supple-
mental Fig. 6A,B). Furthermore, expression of sox18 and
sox7 was normal in mafba
uq4bh
mutants, suggesting that
these genes are upstream of but not downstream from
mafba (Supplemental Fig. 6D,E).
Given that the transgenic overexpression of Vegfc in-
duces proliferation of VECs, we asked whether the reduc-
tion of mafba VEC expression in vegfc-induced/MO-soxF-
injected embryos was concurrent with a reduction in VEC
proliferation. Importantly, Vegfc overexpression in sox7/
sox18 double-knockdown embryos still induced robust
VEC proliferation (Fig. 7D). This observation is strikingly
in line with the fact that mafba also played no role in
vegfc-induced VEC proliferation in this transgenic back-
ground. Finally, we examined sox7 and sox18 expression
by qPCR on embryonic ECs FACS-sorted [using Tg(kdrl:
EGFP)] from MO-vegfc and vegfc-induced embryos at
48 hpf. We found that both were mildly reduced in loss-
of-function scenarios and increased in gain-of-function
Figure 4. A cell-autonomous function for
mafba in lymphatic vascular development. (A)
Transplantation of Tg(fli1a:EGFP)-labeled (green)
and dextran blue-labeled (blue) donor cells into a
Tg(lyve1b:DsRed) (red) host derived from a maf-
ba
uq4bh
in-cross. (A′,A′′) Wild-type ECs were ob-
served to contribute to arteries (AECs), veins
(VECs), and lymphatics (LECs) (white arrows)
in sibling and mutant recipients. The boxed ar-
eas indicate high-power regions (shown at right).
(B) Quantification of endothelial contributions
in A′and A′′. Percentage contributions of a cell
to AECs, VECs, and LECs for all vascular grafted
embryos (n= 11 wild type into sibling embryos
with vascular grafts; n= 13 wild type into mu-
tant embryos with vascular grafts). (C) Trans-
plantation of Tg(lyve1b:DsRed)-labeled (red), Tg
(kdrl:EGFP)-labeled (green), and dextran blue-
labeled (blue) donor cells derived from a maf-
ba
uq4bh
in-cross into an unlabeled wild-type
host. Genotyping confirmed donor identity. (C′)
Sibling ECs contributed to AECs, VECs, and
LECs (arrows). (C′′) Mutant ECs contributed
AECs (n= 12/12 grafts) and VECs (n= 3/12 grafts)
but not LECs. The boxed areas indicate high-
power images (shown at right). (D) Quantifica-
tion of endothelial contributions in C′and C′′.
Percentage contributions to AECs, VECs, and
LECs for all vascular grafts. One mutant graft
was observed with lymphatic vascular morphol-
ogy but expressed Tg(lyve1b:DsRed) and Tg
(kdrl:EGFP), indicative of a differentiation defect (see Supplemental Fig. 3B). Bars, 30 μm.
Mafb in lymphangiogenesis
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scenarios, where normalized expression of endothelial
egfp was not changed (Fig. 7E; Supplemental Fig. 6C).
Discussion
Taken together, the observations above demonstrate that
the transcription factor Mafba is essential for embryon-
ic lymphangiogenesis during zebrafish development. In
mice, MAFB has established roles in directing hindbrain
segmentation and podocyte, pancreatic β-cell, and hema-
topoietic lineage differentiation but no known function
in vascular lineages (Sieweke et al. 1996, 1997; Moens
et al. 1998; Kelly et al. 2000; Sadl et al. 2002; Bakri et al.
2005; Moriguchi et al. 2006; Artner et al. 2007). Our find-
ings of a cell-autonomous role in lymphatic vessel devel-
opment, given the previously described functions in cell
fate and differentiation, suggest a likely role in endothelial
lineage decisions or differentiation. A function in ongoing
LEC differentiation could explain the migration defect ob-
served if critical machinery was not switched on in PLs
after they initially sprout from the PCV. To fully under-
stand how Mafba elicits such a specific phenotype in
LECs, it is clear that the characterization of downstream
genes and pathways is now needed.
Vegfc is the major driver of developmental lymphangio-
genesis in vertebrates (Karkkainen et al. 2004). In zebra-
fish, Vegfc induces VEC proliferation in the PCV (Helker
et al. 2013; Le Guen et al. 2014) and the sprouting of ve-
nous precursors and PLs from the PCV during secondary
angiogenesis (Hogan et al. 2009a; Villefranc et al. 2013).
Our observations of mafba expression and the expression
of a subset of mafba-dependent genes in Vegfc loss-of-
function and gain-of-function embryos indicate that
mafba is responsive to Vegfc signaling during develop-
ment. We showed previously that mutations in genes
within the Vegfc pathway completely block the pheno-
types caused by overexpression of Vegfc (Le Guen et al.
2014), yet here we observed that loss of mafba has no im-
pact on transgene-induced proliferation while being cru-
cial for lymphatic development. This suggests that Vegfc
signaling has multiple downstream outcomes in ECs
Figure 5. mafba-dependent endothelial
genes are Vegfc-dependent, and mafba is
up-regulated by Vegfc signaling. (A) Sche-
matic summary of RNA-seq from FACS-
sorted sibling and mafba
uq4bh
mutants at
48 hpf. n= 23 down-regulated genes; n= 68
up-regulated genes. P< 0.01 using EdgeR
analysis. (B) qPCR validation of RNA-seq.
RNA-seq relative changes and qPCR fold
changes are displayed side by side for a sub-
set of up-regulated and down-regulated
genes. Mean ± SEM. The boxed area con-
tains down-regulated genes. (C) Fifteen
down-regulated genes analyzed by qPCR
in FACS-sorted ECs from 48-hpf vegfc-in-
duced and 30-hpf MO-vegfc embryos. Re-
ductions in expression were observed in
both mafba
uq4bh
and MO-vegfc embryonic
endothelium. Mean ± SEM. (D) Expression
of mafba in control uninjected (n= 30/33),
MO-vegfc-injected (n= 18/18), and MO-
vegfr3-injected (n= 33/33) embryos at 48
hpf. Enriched dorsal PCV expression (ar-
rows) is absent in MO-vegfc and MO-vegfr3
embryos (brackets). Bars, 100 μm. (E) qPCR
validation of mafba reduction in FACS-
sorted ECs from MO-Vegfc embryos. Ex-
pression is given relative to the geometric
average of kdrl,cdh5, and lyve1b expres-
sion. Mean ± SEM. (F) Expression of mafba
in control and vegfc-induced [Tg(prox1a:
Kalt4);Tg(10xUAS:Vegfc)] embryos. mafba
expression is induced throughout the PCV
in vegfc-induced embryos. n= 16/16. ISH
staining was for shorter periods than in E
to highlight increased expression. Bars,
100 μm. (G) qPCR validation of mafba in-
duction in FACS-sorted ECs from Tg
(prox1a:Kalt4);Tg(10xUAS:Vegfc) embryos. Expression is given relative to the geometric average of kdrl,cdh5, and lyve1b expression.
Mean ± SEM.
Koltowska et al.
1624 GENES &DEVELOPMENT
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and that different cellular responses are controlled by dif-
ferent effectors. In line with this observation, the cellular
defect in mafba
uq4bh
mutants occurs later than in vegfc,
vegfr3, or ccbe1 mutants (Hogan et al. 2009a; Villefranc
et al. 2013; Le Guen et al. 2014), with mafba controlling
LEC migration from the horizontal myoseptum rather
than sprouting of LEC precursors from the PCV. It will
be intriguing to discover whether MAFB proteins play spe-
cialized roles regulating a discrete subset of functional
genes rather than broad roles in LEC identity/transcrip-
tion and determine how this compares with other tran-
scription factors, such as PROX1, which is maintained
by VEGFC signaling in mice (Srinivasan et al. 2014).
We further investigated potential mechanisms by
which Vegfc might regulate the levels of mafba in zebra-
fish. In mice, MafB can act together with Ets1, and there
is evidence that Vegfc can modulate Ets-mediated tran-
scription (Sieweke et al. 1996; Yoshimatsu et al. 2011);
however, we examined morpholino knockdown models
and gene expression levels and found no evidence that
Mafba acts coordinately with Ets factors in lymphatic de-
velopment (Supplemental Fig. 6F–J). In mice, the tran-
scription factor Sox18 controls lymphangiogenesis in a
partially redundant manner with other SOXF transcrip-
tion factors in different mouse strains (Francois et al.
2008; Hosking et al. 2009). The transcription factors
Sox18 and Sox7 function redundantly during zebrafish
blood vascular development, with dMO-injected embryos
displaying early arterial–venous defects that are recapitu-
lated in genetic mutants and do not allow for controlled
analysis of later lymphangiogenesis (Cermenati et al.
2008; Herpers et al. 2008; Pendeville et al. 2008; Herm-
kens et al. 2015). Interestingly, recent work has shown
that SOXF transcription factors can be up-regulated and
nuclear-localized in response to VEGF/VEGFC–ERK sig-
naling to control downstream gene expression and vascu-
lar lineage decisions (Deng et al. 2013; Duong et al. 2014).
We investigated whether SOXF transcription factors
could link Vegfc signaling to mafba expression. We found
that Sox18 and Sox7 are necessary for normal mafba ex-
pression and further showed that they indeed mediate
the observed induction of mafba by transgenic overex-
pression of Vegfc. Taken together, these observations led
us to a working model of how this pathway functions to
control lymphatic development (Fig. 7F).
The question of how cells acquire LEC identity and
differentiate has led to the characterization of a number
of transcriptional regulators of LEC fate and differentia-
tion (Wigle and Oliver 1999; Francois et al. 2008; Sriniva-
san et al. 2010; Srinivasan and Oliver 2011). How these
transcription factors combine and integrate as a function-
al regulatory network and how they are controlled by
extrinsic signals to modulate precise spatiotemporally
controlled gene expression remain to be elucidated. The
Figure 6. mafba controls LEC migration and is dispensable for Vegfc transgene-induced proliferation. (A) Representative images from
time lapse of LEC migration from sibling (n= 3) and mafba
uq4bh
mutant (n= 3) embryos. Visualized in Tg(lyve1:DsRed) (white) embryos
from 48 hpf. See Supplemental Movies 1 and 2. (Arrows) LECs. Bars, 20 μm. (B)vegfc-induced embryos display increased VEC numbers
(bottom left panel) compared with control embryos (top left panel). (Right panels) Increased VEC number is still observed in vegfc-induced
mafba
uq4bh
mutant embryos. Tg(fli1a:nlsEGFP) (white) labels endothelial nuclei. Bars, 30 μm. (C) Quantification of VEC number in sib-
ling (n= 4), sibling vegfc-induced (n= 5), mafba
uq4bh
(n= 6), and mafba
uq4bh
vegfc-induced (n= 6). Mean ± SEM; t-test. (ns) No significant
difference.
Mafb in lymphangiogenesis
GENES &DEVELOPMENT 1625
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addition of Mafb as a crucial regulator of lymphangiogen-
esis provides a new direction and increases the complexity
of LEC gene regulation. Importantly, many transcription-
al regulators of LEC development play central roles in
lymphatic vascular diseases (Fang et al. 2000; Finegold
et al. 2001; Irrthum et al. 2003; Ostergaard et al. 2011).
In addition to gaining a deeper mechanistic understanding
of LEC fate acquisition and differentiation, it will be inter-
esting to determine whether MAFB transcription factors
contribute to vascular pathologies in the future.
Materials and methods
Zebrafish
Animal work followed the guidelines of the animal ethics com-
mittee at the University of Queensland. The forward genetic
screen was based on previous studies, with mutagenesis as de-
scribed previously (de Bruijn et al. 2009). The genomic sequenc-
ing pipeline was based on previous studies (Leshchiner et al.
2012) and will be described in full elsewhere. Published zebrafish
lines were Tg(fli1a:nEGFP)
y7
(Lawson and Weinstein 2002), Tg
(-5.2lyve1b:DsRed)
nz101
(Okuda et al. 2012), TgBAC(prox1a:
KalTA4-4xUAS-ADV.E1b:TagRFP)
nim5
(Dunworth et al. 2014;
van Impel et al. 2014), Tg(flt1:YFP)
hu4624
(Hogan et al. 2009a),
and Tg(kdrl:EGFP)
s84 3
(Jin et al. 2005).
Transgenesis, morpholinos, genotyping, and genome editing
10xUAS:vegfc plasmid DNA was generated using the full-length
zebrafish vegfc cDNA cloned into the Gateway pME vector
(pDON-221) using Gateway technology (Hartley et al. 2000). To
generate the Tg(10xUAS:vegfc)
uq2bh
strain, 20 ng/μL plasmid
DNA and 25 ng/μLtol2 transposase mRNA were injected in
one-cell stage embryos, and F1 founders were identified by
Figure 7. mafba is downstream from Vegfc and SoxF transcription factors. (A) Expression of mafba at 30 hpf in control uninjected (n=38/
41) and MO-sox18/sox7 dMO-injected (n= 24/25) embryos (left) and at 48 hpf in control uninjected (n= 30/33) and MO-sox18/sox7 dMO-
injected (n= 18/18) embryos (right). Arrows indicate PCV, and an asteriskindicates reduced expression. Bars, 100 μm. (B)mafba expression
at 48 hpf in control and vegfc-induced embryos as well as vegfc-induced embryos injected with MO-sox7,MO-sox18, and MO-sox18/sox7
dMO. Arrows indicate PCV expression, and asterisks indicate reduced/absent expression. Bars, 100 μm. (C) Quantification of mafba ex-
pression in embryos from E, with embryos scored as high, medium, and low mafba expressors. Control, 88%, n= 37/42, high; MO-sox7,
89%, n= 39/44, high; MO-sox18, 71%, n= 25/35, medium; dMO, 71%, n= 34/48, low. (D) Quantification of VEC number in vegfc-induced
control and MO-sox18/sox7 dMO-injected embryos. Mean ± SEM. Control, n= 12; control vegfc-induced, n= 34; dMO, n= 11; dMO vegfc-
induced, n= 25; t-test. (ns) No significant difference. (E) qPCR for sox18 expression in FACS-sorted zebrafish ECs from MO-vegfc,vegfc-
induced, and control embryos. egfp served as a control for EC expression levels. Expression is relative to the geometric average of kdrl,
cdh5, and lyve1b expression. Mean ± SEM. (F) Working model of Mafbafunction in lymphangiogenesis: Vegfc up-regulates mafba expres-
sion in secondary sprouts and in a Sox18-dependent (redundancy with Sox7) manner. Mafba is essential for PL migration. Vegfc controls
venous proliferation independently of Mafba or Sox18/7.
Koltowska et al.
1626 GENES &DEVELOPMENT
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PCR. mafba full-length cDNA was cloned into the pCS2
+
vector
and used as a template for riboprobe synthesis. MO-vegfr3,MO-
vegfc,MO-sox7, and MO-sox18 were described previously
(Herpers et al. 2008; Le Guen et al. 2014). Two morpholino
against ets1 (Pham et al. 2007) were used as described previously,
and two morpholinos against ets2 were designed and injected
(5 ng per embryo). CRISPR genome editing for mafba was
performed as described in Gagnon et al. (2014) to generate the
mafba
uq5bh
(5-base-pair [bp] del) allele. All primer and morpholino
sequence details are provided in the Supplemental Material.
Whole-mount ISH
Whole-mount ISH was performed as described previously (Karto-
pawiro et al. 2014) using the mafba probe (see above) generated
from plasmid linearized using ClaI. Probes for vegfc (Ober et al.
2004), vegfr3 (Hogan et al. 2009b), cxcr4a and cxcl12b (Coxam
et al. 2014), efnb2a (Durbin et al. 1998), ephb4a (Cooke et al.
1997), and sox18 and sox7 (Herpers et al. 2008) were used as pre-
viously described.
FACS and gene expression analysis
Isolation of zebrafish embryonic ECs, RNA extraction, cDNA
preparation, and qPCR were performed as described previously
(Coxam et al. 2014; Kartopawiro et al. 2014). For isolating cells
from sibling and mafba
uq4bh
mutants, embryos were divided
into the phenotypic categories based on the otic phenotype at
48 hpf. For isolating cells from vegfc-induced and siblings, embry-
os were divided into phenotypic categories based on vascular phe-
notype at 48 hpf. For MO-vegfc and uninjected control, cells were
isolated at 30 hpf and 48 hpf. Primer sequences are in the Supple-
mental Material. RNA-seq was performed using the Illumina
NextSeq500 and generated average 76-bp reads, which were
then mapped to the reference genome (danRer7/Zv9). Full details
on library preparation, sequencing, and analysis are in the Supple-
mental Material.
Immunohistochemistry
Immunohistochemistry for anti-GFP was performed according to
the following protocol. Embryos were fixed in 4% PFA (parafor-
maldehyde) overnight and washed five times with PBST (0.1%
Tween in PBS [phosphate buffered saline]). Embryos were blocked
in PBDT (PBS with 1% BSA, 1% DMSO, 0.1% Triton-100) with
10% horse serum for 3 h, anti-GFP (chicken polyclonal to GFP,
1:200; ab13970) was added, and embryos were incubated over-
night. Embryos were washed five times for 30 min in PBDT and
then incubated in PBDT and 10% horse serum with secondary an-
tibody (goat anti-chicken IgG, 1:400; Alexa 488; Invitrogen,
A11039) and DAPI (1:1000; Sigma Aldrich) overnight. Embryos
were washed five times for 30 min in PBST and imaged.
Transplantation
Transplantation was performed essentially as described previ-
ously (Hogan et al. 2009a) with the following changes. Donor em-
bryos were injected with dextran cascade blue (10,000 MW,
Invitrogen) at 5 ng/nL (1 nL per embryo). Cells from wild-type
donor embryos [Tg(fli1a:EGFP)] were transplanted into host
embryos derived from mafba heterozygous in-crosses [Tg
(-5.2lyve1b:DsRed)]. For reciprocal transplants, mutant cells [Tg
(kdrl:EGFP);Tg(-5.2lyve1b:DsRed)] were transplanted into unla-
beled wild-type hosts, and donors were genotyped. Embryos
with successfully transplanted ECs were cultured until 5 dpf.
Imaging and quantification
Live and fixed embryos were mounted laterallyand imaged using
a Zeiss LSM 710 FCS confocal microscope. All images were pro-
cessed using either ImarisX64 7.70 and/or ImageJ 1.47 (National
Institutes of Health) software. The number of LEC nuclei [ex-
pressing Tg(fli1a:nEGFP)] coexpressing Tg(-5.2lyve1b:DsRed)
across five somites through a Z-stack was manually counted
using ImageJ 1.47 (National Institutes of Health) software
(Fig. 1A,C,D–G; Supplemental Fig. 1C) The number of Prox1-
positive LECs expressing Tg(prox1aBAC:KalTA4-4xUAS-E1b:
uncTagRFP)
nim5
;Tg(10xUAS:Venus) detected by α-GFP in green
and costained with DAPI (to label nuclei) across five somites
was manually counted using ImageJ 1.47 (National Institutes
of Health) software (Fig. 1H,I). For the quantification of EC num-
bers shown in Figure 5, the spot tool in ImarisX64 7.70 software
was used. For each sample, the Tg(fli1a:nEGFP) nuclei were se-
lected based on fluorescence intensity, and the total number of
GFP-positive nuclei (across five somites) in a full Z-stack was
calculated.
Acknowledgments
Imaging was performed in the Australian Cancer Research Foun-
dation’s Dynamic Imaging Facility at the Institute for Molecular
Bioscience. Sequencing was performed by the Institute for Molec-
ular Bioscience Sequencing Facility. K.K. was supported by a
Lymphatic Education and Research Network Post-doctoral Fel-
lowship, A.K.L. was supported by a University of Queensland
Post-doctoral Fellowship, B.M.H. was supported in part by an
Australian Research Council Future Fellowship (FT100100165)
and in part by a National Health and Medical Research Coun-
cil/National Heart Foundation Career Development Fellowship
(1083811), M.F. was supported by a National Health and Medical
Research Council Career Development Fellowship (1011242),
and K.A.S. was supported by an Australian Research Council Fu-
ture Fellowship (FT110100496). J.W.A. and P.S.C. receive funding
from The Ministry of Business, Innovation, and Employment; the
Health Research Council of New Zealand; and the Auckland
Medical Research Council. This research was supported by Na-
tional Health and Medical Research Council grant 1050138 and
the Cariplo Foundation.
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essential for embryonic lymphangiogenesis in zebrafish
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