Genomes & Developmental Control
Combinatorial function of ETS transcription factors
in the developing vasculature
Van N. Phama, Nathan D. Lawson1, Joshua W. Mugford2, Louis Dyeb, Daniel Castranovaa,
Brigid Loa, Brant M. Weinsteina,⁎
aLaboratory of Molecular Genetics, NICHD, NIH, Building 6B, Room 309, 6 Center Drive, Bethesda, MD 20892, USA
bMicroscopy and Imaging Core, NICHD, NIH, Bethesda, MD, USA
Received for publication 12 September 2006; revised 17 October 2006; accepted 20 October 2006
Available online 25 October 2006
Members of the ETS family of transcription factors are among the first genes expressed in the developing vasculature, but loss-of-function
experiments for individual ETS factors in mice have not uncovered important early functional roles for these genes. However, multiple ETS
factors are expressed in spatially and temporally overlapping patterns in the developing vasculature, suggesting possible functional overlap.
We have taken a comprehensive approach to exploring the function of these factors during vascular development by employing the genetic
and experimental tools available in the zebrafish to analyze four ETS family members expressed together in the zebrafish vasculature; fli1,
fli1b, ets1, and etsrp. We isolated and characterized an ENU-induced mutant with defects in trunk angiogenesis and positionally cloned the
defective gene from this mutant, etsrp. Using the etsrp morpholinos targeting each of the four genes, we show that the four ETS factors
function combinatorially during vascular and hematopoietic development. Reduction of etsrp or any of the other genes alone results in either
partial or no defects in endothelial differentiation, while combined reduction in the function of all four genes causes dramatic loss of
endothelial cells. Our results demonstrate that combinatorial ETS factor function is essential for early endothelial specification and
© 2006 Elsevier Inc. All rights reserved.
Keywords: Zebrafish; ETS transcription factors; Intersegmental vessels; Vascular development; Angiogenesis
The ETS factors are a large family of transcriptional
regulatory proteins involved in a wide variety of developmental
and postnatal processes, in an equally diverse array of tissues.
The first known ETS family member was the proto-oncogene
Ets1, the cellular progenitor of v-ets, a viral oncogene found in
the genome of the E26 acute leukemia retrovirus (Leprince et
al., 1983; Nunn et al., 1983). More than 50 additional members
of this family have now been identified, all of which share an 85
amino acid conserved DNA binding domain, the “ETS
domain,” a winged helix-turn-helix motif generally located in
the C-terminal half of the protein. The ETS domain binds to
DNA sequence consisting of a core GGAA/T motif. Most ETS
family members are transcriptional activators except for a few
(Erf, Net, Tel) that have been shown to have repressor activity.
The activity of ETS transcription factors is regulated through
their interaction with a large number of different structurally
unrelated transcription factors such as AP1, MafB, and CBP
(for a comprehensive list, see Lelievre et al., 2001; Verger and
Recent studies have suggested that a number of different
ETS factors play important roles in hematopoietic and
vascular development during early embryogenesis, although
most in vivo loss-of-function data have not provided
compelling evidence for essential roles for these genes in
the specification or differentiation of these tissues (reviewed in
Lelievre et al., 2001; Sato, 2001). During murine develop-
ment, the Ets1 gene is initially expressed broadly in ventral
Developmental Biology 303 (2007) 772–783
E-mail address: email@example.com (B.M. Weinstein).
1Current address: U. Massachusetts, Worcester, MA, USA.
2Current address: Harvard University, Cambridge, MA, USA.
0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
mesoderm, becoming progressively restricted to hemangio-
genic mesoderm and then endothelium (Pardanaud and
Dieterlen-Lievre, 1993; Queva et al., 1993). Antisense
oligonucleotides against Ets1 inhibit angiogenesis in the
chick chorioallantoic membrane (CAM) assay (Wernert et
al., 1999), and dominant-negative Ets1 inhibits angiogenesis
in endothelial cells in vitro and in implanted matrigel plugs
(Nakano et al., 2000), all supporting the idea that this factor
plays an important role in angiogenesis. However, Ets1 null
mice are viable and fertile and have no detectable vascular
defects (Barton et al., 1998; Bories et al., 1995; Muthusamy et
al., 1995). In zebrafish, however, a recent study reported that
morpholino knockdown of the novel ets1 related protein etsrp
does cause defects in blood vessel formation (Sumanas and
Like Ets1, the Fli1 and closely related ERG (Ets-related)
genes become progressively restricted to endothelial cells in
the trunk during zebrafish and Xenopus development (Brown
et al., 2000; Meyer et al., 1995). Overexpression of either Fli1
or ERG by microinjection into Xenopus laevis embryos
causes ectopic endothelial differentiation, in addition to other
defects (Baltzinger et al., 1999; Remy et al., 1996). However,
transgenic mice overexpressing Fli1 in a variety of tissues
under H-2Kk promoter control do not exhibit vascular
abnormalities, although they do develop a lethal renal
immunologic disease (Zhang et al., 1995). Furthermore,
mice homozygous for a targeted disruption of Fli1 form a
functional network of blood vessels, indicating that vasculo-
genesis and angiogenesis proceed normally, although they
develop CNS hemorrhage (Hart et al., 2000; Spyropoulos et
al., 2000). Mice with targeted disruption of the TEL repressor
have normal vasculogenesis, with histologically normal dorsal
aorta, intersomitic vessels and head veins at E9.5. However,
they exhibit defective yolk sac angiogenic remodeling as well
as intra-embryonic apoptosis of mesenchymal and neural cells
(Wang et al., 1997). It has been suggested that Tel functions in
the maintenance of the developing vascular network rather in
the specification, differentiation, or proliferation of endothelial
The early and specific expression of ETS factors in
vascular tissues and their mesodermal progenitors has led to
speculation that these factors might be important in the
establishment of this lineage and the differentiation of
angioblasts and endothelial cells, but as noted above in vivo
evidence for this has been difficult to obtain from loss-of-
function experiments. It is likely that a significant difficulty in
probing the functional roles of ETS factors in the vasculature
is the extensive overlap between these factors in this tissue,
making it difficult to perform effective loss-of-ETS-function
experiments. We have taken a comprehensive approach to
exploring ETS factor function in the vasculature by using the
zebrafish to simultaneously reduce the levels of multiple ETS
family members. We identified four different vascular ETS
factors in the zebrafish and characterized a genetic mutant in
one of these genes, etsrp. We show that the four genes
function cooperatively in the differentiation and maintenance
Materials and methods
Zebrafish (Danio rerio) embryos were obtained and raised and fish
maintained as described (Kimmel et al., 1995; Westerfield, 1995). The Tg
(fli1:EGFP)y1transgenic zebrafish line was described previously (Lawson and
Weinstein, 2002). An ENU F3 genetic screen was performed using this line to
isolate the etsrpy11mutant (Lawson et al., unpublished results). Embryos imaged
post-1.5 dpf were treated with 1-phenyl-2-thiourea (PTU) to inhibit pigment
formation (Westerfield, 1995).
Meiotic and physical mapping of the y11 mutation
Meiotic and physical mapping was performed essentially as described
previously (Roman et al., 2002) using an EK Tg(fli1:egfp)y1; y11/TL
polymorphic mapping cross. Bulked segregant analysis was performed
using a 192-marker panel of CA repeat markers (the list of markers in this
panel is available upon request). Oligonucleotide sequences for the markers
noted in Fig. 2 are available at http://zebrafish.mgh.harvard.edu/zebrafish/
ssrQuery.aspx. BAC clones were identified by screening the CHORI 211
Zebrafish BAC library filters (Children's Hospital Oakland Research Institute)
using32P labeled oligonucleotide probes. PAC clones were identified from
DNA pools by PCR as described by the supplier (Chris Amemiya Lab). BAC
and PAC DNAs were prepared using Nucleobond columns (Clontech). BAC
end sequences were obtained from http://trace.ensembl.org/perl/ssahaview?
server=danio_rerio. PAC end sequences were determined by sequencing.
Additional genomic sequences were obtained through SSAHA2 searches of
trace data from the Sanger Institute (http://www.ensembl.org/Danio_rerio/
blastview). PCR primers designed against non-repetitive regions of PAC and
BAC ends and genomic sequences were used to establish physical contigs and
look for polymorphisms for use in meiotic mapping. To identify polymor-
phisms, PCR products amplified from genomic DNA from an individual wild-
type, heterozygous, and mutant embryos (genotyped based on flanking
markers) were sequenced. SNPs were assayed as RFLPs, when possible, or
were converted to RFLPs using derived cleaved amplified polymorphic
sequence (dCAPS) analysis using a dCAPS Finder program (Neff et al.,
Cloning of full-length zebrafish etsrp, fli1b, and ets1 cDNAs
Sequences encompassing the 5′ UTR to 3′ UTR of etsrp were obtained
using available zebrafish EST (GenBank #s AI877585, AL915831, AI 793509,
AI793542) and genomic trace sequences. 5′ RACE was performed from 24 hpf
cDNA to obtain the complete 5′ UTR sequence. The full-length cDNA of etsrp
was obtained by high fidelity PCR on 24 hpf cDNA using PfuUltra High-
Fidelity DNA polymerase (Stratagene) and the primers 5′-CTTTAAGATATG-
GAAATGTACCAATCGG-3′ and 5′-CCAATCCTTCGATTCCTCCTCTA-3′.
A single product was obtained, cloned into pCRII-TOPO (Invitrogen), and
verified by sequencing.
The 3′ end of fli1b was obtained using available zebrafish EST sequence
(GenBank # CA472045). 5′ RACE was performed from 24 hpf cDNA to
obtain the complete 5′ UTR and coding sequence. The full-length coding
sequence of zebrafish fli1b was obtained by high fidelity PCR as above with
primers 5′-CAGAAATCTGCAATGGACT-3′ and 5′-GTGACTGTTTTAA-
TAATAAGTGTTC-3′. A single product was obtained, cloned into pCRII-
TOPO (Invitrogen), and verified by sequencing.
The full-length coding sequence of zebrafish ets (GenBank # BC092935)
was amplified by high fidelity PCR as above using primers 5′-ACAGACTCTG-
TACGTTTGAATGCGT-3′ and 5′-GTCCAGACTTTACTCGTCCGTGTC-3′.
A single product was obtained, cloned into pCRII-TOPO (Invitrogen), and
verified by sequencing.
773V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
Expression constructs, in vitro transcription/translation, and RNA
The etsrp pCRII-TOPO clones described above were used as templates in a
PCR reaction with PfuUltra High-Fidelity DNA polymerase (Strategene) and
primers extended with a BamHI site (5′-AGGGATCCCAGAGCTGAGG-
CACTGTCAAAACC 3′) and an XbaI site (5′-GCTCTAGACCAATCCTTC-
GATTCCTCCTCTA-3′). The resulting PCR product was digested with BamHI
and XbaI and cloned into Bam/XbaI-digested pCS2+. The construct was
verified by sequencing.
A full-length fli1 cDNA clone (GenBank NM131348) was ordered (Open
Biosystems, Inc.) and used as template in a PCR reaction with PfuUltra High-
Fidelity DNA polymerase (Strategene) and primers extended with an EcoRI site
(5′-CGGAATTCTAATTCAGACGCGTGTCATGT-3′) and an XhoI site (5′-
CCGCTCGAGCCATCTTCGAGTGCAGTTCAAG-3′). The resulting PCR
product was digested with EcoRI and XhoI and cloned into EcoRI/XhoI
digested pCS2+. The construct was verified by sequencing.
pCS2+/ets1 and pCS2+/fli1b
The ets1 pCRII-TOPO and fli1b pCRII-TOPO clones described above were
digested with BamHI and XbaI and cloned into BamHI/XbaI digested pCS2+.
The etsrp pCRII-TOPO clones described above were used as templates in a
PCR reaction with PfuUltra High-Fidelity DNA polymerase (Strategene) and
primers 5′-GCCACCATGGAAATGTACCAATCTGG-3′ and 5′-ATGTGTC-
CAGGACTCTGTGGTTTCCT-3′. A single product was obtained, cloned into
pcDNA3 (Invitrogen), and verified by sequencing. The etsrp pcDNA3.1
construct was used as template in a PCR reaction with PfuUltra High-Fidelity
DNA polymerase (Strategene) and primers extended with an NheI site (5′-
CTAGCTAGCTAGGCCACCATGGAAATGTACCAA-3′) and an AscI site (5′-
TTGGCGCGCCAAACTCAGACAATGCGATGC-3′). The resulting PCR
product was digested with NheI and AscI and cloned into NheI/AscI-digested
pFliL. The construct was verified by sequencing.
Capped mRNA for injection was synthesized from NotI-digested pCS2
expression plasmids using the mMessage mMachine SP6 kit according to the
provider's protocol (Ambion). Capped mRNA was injected into 1- to 4-cell
embryos either into a single blastomere or into the streaming yolk cytoplasm,
just beneath the blastomeres, using a pneumatic picopump and micromanipu-
lator(World PrecisionInstruments), as describedpreviously(Westerfield, 1995).
In vitro transcription/translation assays were performed using the TNT SP6
Quick Coupled Transcription Translation System (Promega), following the
included: a standard control morpholino (5′-CCTCTTACCTCAGTTACAATT-
TATA-3′); etsrp (5′-GGTTTTGACAGTGCCTCAGCTCTGC-3′) targeting −10
to −34 of the 5′ UTR of etsrp; etsrpE/I-2 (5′ AAATAAGATATTACCATAT-
GAACTG 3′) targeting the exon 2/intron 2 boundary; etsrpE/I-3 (5′-TGA-
GATGCTCACCTTTGTGCAACAG-3′) targeting the exon 3/intron 3 boundary;
ets1 (5′-GTCATGGTCACGCATTCAAACGTAC-3′) targeting −10 to +15 of the
5′ UTR and coding region of ets1; fli1b (5′-GGTTAAACTTGAGCTATG-
TAAACCC 3′) targeting −57 to −81 of the 5′ UTR of fli1b; fli1 (5′-
GTTCCGTCCATTTTCCGCAATTTTC-3′) targeting −14 to +11 of the 5′ UTR
text): etsrp 2 ng;etsrpE/I-2, 15 ng;etsrpE/I-3, 12 ng;combinedetsrpE/I-2+etsrpE/
I-3, 10 ng each (the combination gives a stronger phenotype similar to the “etsrp”
morpholino, theindividual“etsrpE/I”morpholinosalsogive similar butlesssevere
phenotypes); ets1, 2 ng, fli1b, 15 ng; fli1, 15 ng. “Medium” and “low” doses of
morpholinos used in this study were one-half and one-quarter of the “high” dose,
respectively. All morpholinos used in this study were shown to specifically and
strongly reduce the levels of the targeted gene product in an in vitro transcription–
translation assay without affecting the levels of the other three ETS factors
(Supplemental Fig. 1). Morpholino injections were performed on 1- to 4-cell
embryos as described previously (Lawson et al., 2002).
Whole-mount in situ hybridization and TUNEL staining
Antisense mRNA probes for fli1, flt4, vecdn, flk1, plxnd1, efnb2, gata1,
and gata2 were prepared as described (Brown et al., 2000; Detrich et al., 1995;
Larson et al., 2004; Lawson et al., 2001; Thompson et al., 1998; Torres-Vazquez
et al., 2004). To derive etsrp, fli1b, and ets1 riboprobes, the corresponding
pCRII-TOPO constructs were linearized with XhoI, BamHI, or SpeI,
respectively, and transcribed using T7 RNA polymerase. Whole-mount in situ
hybridization was performed essentially as described elsewhere (Hauptmann
andGerster, 1994). TUNEL stainingof 24hpf zebrafishembryoswas performed
as described previously (Parng et al., 2004).
Microscopic imaging methods
Transmitted light images were obtained with a Leica MZ12 microscope
equipped with a ProgRes mF digital camera (Jenoptik, Eching, Germany). Two-
photon microscopy of Tg(fli1:EGFP)y1zebrafish embryos and larvae was
performed using a Radiance 2000 imaging system (BioRad) with 960 nm pulsed
mode-locked laser emission from a tunable Ti-Sapphire laser (Tsunami laser,
Spectra Physics Inc.). Stacks of frame-averaged (5 frames) multiphoton optical
slices were collected digitally, and 2-D or 3-D reconstructions of image data
were prepared using the LaserSharp (BioRad) or Metamorph (Universal
Imaging) software packages. The fluorescence images shown in this paper are
single-view 2-D reconstructions of collected image Z-series stacks, recon-
structed at an angle of 0°.
For transmission electron microscopy, zebrafish embryos were fixed at room
temperature in buffered 2.5% glutaraldehyde (pH 7.3), post-fixed in 2% osmium
tetroxide, and processed into Spurr's epoxy via increasing concentrations of
ethanol followed by propylene oxide. Semithin 1 μm plastic sections were cut
from at least six plastic blocks and stained with Toluidine Blue O stain. Thin
sections of at least two blocks were prepared and stained with uranyl acetate and
lead citrate and examined in a JEOL 1010 electron microscope operating at
80 kV. Anesthetized adult zebrafish was cut transversely into 6 parts and fixed at
4°C in 2.5 glutaraldehyde–2% paraformaldehyde–0.1 M phosphate buffer (pH
7.4) for 2 h, post-fixed at 42°C in 1% osmium tetroxide–0.1 M phosphate buffer
(pH 7.4) for 2 h, and processed into Agar 100 resin (Agar Scientific Limited,
UK) via increasing concentrations of ethanol followed by QY-2. Semithin
0.5 μm plastic sections were cut and stained with Toluidine Blue O stain. Thin
sections were stained with uranyl acetate and lead citrate and examined in a
Hitachi H-7100 electron microscope operating at 100 kV.
Identification of y11, a mutant defective in vascular
morphogenesis and angiogenesis
We identified the y11 mutant in an F2 haploid screen for
mutants with trunk vascular defects carried out in the Tg
(fli1:EGFP)y1transgenic background (Lawson and Weinstein,
2002 and unpublished results). Homozygous y11 mutants
have defects in both vascular morphogenesis and angiogen-
esis. Mutants have a beating heart, but they lack trunk
circulation at 24 hpf and 48 hpf. No reduction in initial
numbers of EGFP-positive cells is apparent in the trunk at
24 hpf, suggesting that there is no defect in specification of
angioblasts during initial vasculogenesis (data not shown).
However, increased TUNEL staining is detected in the axial
774V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
vessels of y11 mutants at 24 hpf (Figs. 1A, B), and the
primary vasculogenic vessels (including the trunk dorsal
aorta and posterior cardinal vein) fail to undergo proper
morphogenesis to form defined tubular vessels (Figs. 1C, D,
F, G). Transmission electron microscopy reveals that mutant
angioblasts fail to form an ordered epithelium and appear
disorganized (Figs. 1J, K). Angiogenic intersegmental vessel
sprouts are absent at 24 hpf in mutant trunks (Figs. 1C, D),
but aberrant stunted sprouts appear by 48 hpf (Figs. 1H, I).
Mutant intersegmental vessel sprouts fail to lumenize
properly and form inappropriate branches (Figs. 1H, I),
although the overall patterning of the trunk vasculature is not
grossly perturbed as it is in vascular patterning mutants such
as out of bounds (Torres-Vazquez et al., 2004). Inspection of
y11 mutants under transmitted light reveals no developmental
delay or detectable deficits in the patterning, size, or shape
of non-vascular tissues including the somites, notochord,
brain, neural tube, and gut (data not shown). These results
indicate that the function of y11 is restricted to the
Molecular cloning of etsrp, the defective gene in y11 mutants
To identify the defective gene in y11 mutants, we performed
meiotic mapping to localize y11 in the zebrafish genome. Initial
bulked segregant mapping placed y11 on LG16, and fine
genetic mapping using a panel of 1445 mutant embryos from a
polymorphic mapping cross localized y11 to a 0.5 cM interval
between markers z5298 and z624 (Fig. 2A). Chromosome
walks initiated from both markers refined this to a 270 kb,
0.35 cM critical interval defined by 2 recombinants on the
z5298 end and 3 recombinants on the z6240 end. This interval
included the acsI, pycard, etsrp, fli1b, and fbl genes, as well as
genes with homology to mammalian DYRK, ZPdomain, and
GIRK2. The complete coding sequence of each of these genes
was obtained from cDNA from y11 mutants and their wild-type
siblings. Comparison of wild-type and mutant sequences for
each of the genes showed that only one gene has a sequence
alteration resulting in a change in the protein coding sequence in
y11 mutants, the ETS-related gene etsrp. In y11 mutants, a G is
inserted after nucleotide 269 of the etsrp coding sequence,
Fig. 1. y11 mutants have defects in angiogenesis and vascular morphogenesis. (A, B) TUNEL staining of the trunks of 24 hpf wild-type (A) and y11 mutant (B)
animals, low background in wild-type animals and substantially increased staining in the developing axial vessels of y11 mutants (arrows). (C–I) Two-photon
images of Tg(fli1:EGFP)y1transgenic wild-type or y11 mutant animals at 24 hpf (C–G) and 48 hpf (H, I). Wild-type siblings (C, F, H) have intersegmental vessel
sprouts (arrows in panel C) and properly lumenized axial vessels (arrowheads in panel F) at 24 hpf. Intersegmental vessels have formed a lumenized, functional
vascular network by 48 hpf (H). In y11 mutants (D, G, I), intersegmental vessel sprouts are absent at 24 hpf (D) and axial vessels fail to undergo proper tubular
morphogenesis (G). Intersegmental vessels are similarly absent in etsrp morphants at 24 hpf (E). By 48 hpf, y11 mutants have aberrant intersegmental vessel
sprouts that are not fully extended and have some branching and pathfinding errors (I). (J, K) Electron microscopy of 24 hpf zebrafish shows defects in vascular
morphogenesis in y11 mutants. Wild-type siblings have normal single-cell thick endothelial epithelium (arrows) around the dorsal aorta (J), but endothelial cells in
y11 mutants do not form a proper epithelium (K). Anterior is to the left, dorsal up in all panels. Rostral is to the left and dorsal is up in panels A–I. Scale
bars=200 μm (A, B), 200 μm (C–E), 50 μm (F, G), 100 μm (H, I), 10 μm (J, K).
775V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
resulting in +1 frame shift (Fig. 2B). The y11 mutant
polypeptide goes out of frame after amino acid 90, continuing
out-of-frame for an additional 62 amino acids before terminat-
ing. The predicted polypeptide lacks 276 amino acids or
approximately three-fourths of the wild-type etsrp protein,
including the conserved ETS DNA binding domain shown to be
essential for the function of other ETS genes (Fig. 2C). The
etsrp gene is most closely related to the ets1 gene implicated
in vascular and hematopoietic development in other species
(Fig. 2D). We performed in situ hybridization to determine the
expression pattern of etsrp and found that its expression is
restricted to blood vessels and cells within them (see below),
further supporting a function for this gene in vascular and
hematopoietic development. To confirm that the defect in the
etsrp gene is indeed responsible for the y11 mutant phenotype,
we injected morpholino antisense oligonucleotides either
targeting the translation initiation site or a combination of two
morpholinos targeting the first plus second splice donor sites of
the etsrp gene into wild-type Tg(fli1:EGFP)y1embryos. The
translation initiation site (“ATG”) morpholino fully phenocop-
ied the trunk circulation and intersegmental vessel sprouting
defects of y11 mutants (Figs. 1D, E). The combined splice site
morpholino-injected animals gave similar but less dramatic
vascular defects than y11 mutants or the ATG morpholino (data
not shown), so the ATG morpholino was used for all further
We examined whether wild-type etsrp gene product could
support endothelial differentiation. Injection of wild-type etsrp
mRNA into either y11 mutants or wild-type animals at 100 pg
doses resulted in early lethality during gastrulation. However,
Fig. 2. Positional cloning of etsrp, the defective gene in y11 mutants. (A) Genetic and physical map of the y11 interval. Genetic map is shown at top, with the number
of recombinants in approximately 2000 meioses noted in brown (and whether recombinants are on the right or left sides, in parentheses). BAC and PAC clones are
shownas large gray bars, genomicsequence contigs and traces are shownas black bars. The red bar shows the y11 critical interval containingthe entire etsrp transcript.
(B) In y11 mutants, a G is inserted after nucleotide 269 in the etsrp coding sequence. (C) The y11 insertion mutation results in an altered reading frame after amino acid
90 of etsrp coding for an additional novel 62 amino acids (shown in red) before terminating at a stop codon. The y11 mutant protein lacks the highly conserved ETS
DNA binding domain (shown in green in the wild-type protein). See Results and Materials and methods for additional details. (D) ETS family members expressed in
the zebrafish vasculature and related mammalian orthologs. Dendogram of nucleotide similarity across the entire coding region of zebrafish ETS family members
expressed in the zebrafish vasculature and the most closely related mammalian genes. The zebrafish ets1 and fli1 genes are more closely related to their mammalian
counterparts than to other zebrafish ETS family members. The etsrp and fli1b genes are most closely related to ets1 and fli1 genes, respectively.
776 V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
when Tg(fli1:EGFP)y1or Tg(flk1:GFP) animals were injected
at the 4- to 8-cell stage with lower doses (20 pg) of etsrp mRNA
directed to only 1 or 2 cells, most embryos developed to 24 hpf
and older with few abnormalities. Broad ectopic expression of
EGFP-positive cells could be seen in most Tg(fli1:EGFP)y1
animals during mid-somitogenesis (Fig. 3B), with more limited
ectopic expression of GFP noted in a smaller number of Tg
(flk1:GFP) animals (Fig. 3D). Expression of the transgenes was
greatly reduced or extinguished by 48 hpf, however, and no
ectopic blood vessels were noted (data not shown). These
results indicate that the etsrp gene product can initiate
endothelial differentiation, as noted for overexpression of ETS
factors in X. laevis (Baltzinger et al., 1999; Remy et al., 1996).
To test whether endothelial-specific expression of etsrp
could rescue the y11 mutant phenotype, we constructed a vector
in which the fli1 promoter drives etsrp expression. We injected
either fli1-etsrp DNA or control fli1-mRFP1 DNA into embryos
from an incross of y11/+, Tg(fli1:EGFP)y1animals, scoring for
the presence or absence of intersegmental vessel sprouts at
24 hpf. As expected, 25% (8/33 in 2 separate experiments) of
control fli1-mRFP1 injected animals lacked all intersegmental
vessel sprouts at 24 hpf. In contrast, 100% (137/137 in 2
separate experiments) of fli1-etsrp injected animals had
intersegmental vessel sprouts, although some spouts were
missing or less well developed in some animals, as would be
expected from the mosaic expression from injected DNA. This
shows that wild-type etsrp expressed in endothelium can at
least partially rescue the phenotype of y11 mutants. Together, all
of these results indicate that the defective gene responsible for
the y11 mutant phenotype is the ETS family member etsrp, and
the mutation is thus designated etsrpy11.
Multiple ETS family members are expressed in the zebrafish
The functional role of ETS transcription factors in blood
vessel formation has not been fully explored in mice and other
species due to the expression of multiple ETS factors within the
vasculature. We identified and obtained full-length cDNA
sequences for four zebrafish ETS family members with
vascular-restricted expression during early development—two
Fli1 related genes, fli1 and fli1b, and two Ets1 related genes,
ets1 and etsrp. Extensive database searches and analysis by in
situ hybridization of a number of other ETS-related candidate
genes failed to reveal additional genes with vascular expression
(data not shown). The sequence similarity between these genes
and related mammalian ETS family members is shown in Fig.
2D. All four genes begin to be expressed during early
somitogenesis in the trunk lateral mesoderm (Figs. 4A–D).
Expression of ETS genes in vascular progenitors precedes the
expression of vascular markers such as vecdn1 (ve-cadherin)
and flk1 (Fouquet et al., 1997; Larson et al., 2004; Liao et al.,
1997), which are not detected at the 5 somite stage (data not
shown), butare apparent by the10 somite stage (Figs. 4I,J). The
etsrp and fli1 genes both show robust expression at the 5 somite
stage (Figs. 4A, C), although rostral expression of fli1 is not
apparent until the 10 somite stage (Fig. 4G). At 24 hpf, all four
ETS family genes are expressed in the vasculature (Figs. 4Q–T,
W–Z), as is evident from comparison to the expression patterns
of vascular markers flk1 and vecdn1 (Figs. 4U, V, A′, B′). All
four ETS genes are expressed in both the vasculogenic axial
vessels (dorsal aorta and posterior cardinal vein) and in the
angiogenic intersegmental vessel sprouts (Figs. 4W–Z). The
overlapping expression of four different related ETS genes in
developing zebrafish blood vessels suggests that these genes
might have overlapping roles in the vasculature.
etsrpy11mutants have defects in vascular and hematopoietic
To further probe the nature of the vascular defects in etsrpy11
mutants, we compared the expression of a variety of vascular
and hematopoietic genes in wild-type and mutant embryos,
including all four ETS family members. Whole-mount in situ
hybridization with fli1, fli1b, ets1, and etsrp showed no
apparent difference in expression levels or pattern at the 10
somite stage (data not shown). However, expression of a
number of endothelial-specific markers was already affected at
this stage of development in etsrpy11. flk1, flt4 (Thompson et
al., 1998), vecdn1, and plxnD1 (Torres-Vazquez et al., 2004)
expression levels were all reduced at the 10 somite stage, the
earliest time point examined (Fig. 5). By 24 hpf, trunk axial
vessel expression of fli1b and etsrp was strongly reduced (Figs.
6B, D), fli1 appeared slightly reduced (Fig. 6A), while ets1
appeared unchanged (Fig. 6C). These results suggest that etsrp
is not required for the initial expression of ETS genes within
Fig. 3. Induction of ectopic fli1-EGFP and flk1-GFP expression by etsrp
mRNA injection. (A–D) Confocal images of Tg(fli1:EGFP)y1(A, B) and Tg
(flk1:GFP) (C, D) transgenic animals at approximately 14 somite stage/16 h
post-fertilization. (A, C) Uninjected control animals. (B, D) Animals injected at
the 4- to 8-cell stage with 20 pg etsrp mRNA, targeting a limited number of
blastomeres. Ectopic expression of the fli1-EGFP and flk1-GFP transgenes is
noted by large arrows; small arrows show normal expression domains. Rostral is
to the left and dorsal is up in all panels. Scale bar=400 μm.
777 V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
angioblast progenitors, but is required to maintain expression of
both fli1b and its own transcript. Expression of the four
endothelial marker genes was still substantially reduced,
although clearly detectable, within the vasculature of 24 hpf
animals (Figs. 6E–H). The arterial vascular marker efnb2
(Lawson et al., 2001) was also reduced in the dorsal aorta but
not in non-vascular tissues at 24 hpf (Fig. 6I). Thus, etsrp
appears to be required for proper expression of markers of
differentiated endothelial cell fate.
Since ETS genes play a role in hematopoietic development
in other species (Bartel et al., 2000; Barton et al., 1998;
Maroulakou and Bowe, 2000; Remy and Baltzinger, 2000;
Spyropoulos et al., 2000), we also examined the expression of
two markers of the hematopoietic lineage in the zebrafish,
gata1 and gata2 (Detrich et al., 1995). The gata1 gene has been
shown to be critical for erythroid development in zebrafish
(Lyons et al., 2002) and other species. The gata2 gene is
important for hematopoietic stem cell production and expansion
in mice (Ling et al., 2004), and loss of zebrafish gata2 results in
modest decreases in erythroid and myeloid marker expression
(Galloway et al., 2005). Whole-mount in situ hybridization at
the 10 somite stage with gata1 and gata2 probes revealed no
apparent difference in the pattern or level of expression of either
gene between mutant and wild-type animals (data not shown).
By 24 hpf, however, gata1 and gata2 expression was strongly
reduced (Figs. 6J, K). These results suggest that similarly to
ets1 and etsrp expression, maintenance but not initial induction
of hematopoietic gene expression requires etsrp function.
Functional overlap between ETS family members in the
Since we identified four different ETS-related family
members expressed within the vasculature, it seemed likely
that these genes might show functional redundancy and that the
phenotype of loss of etsrp function in etsrpy11mutants might
reflect a partial loss of ETS transcription factor function within
the vasculature. In order to probe the vascular role of ETS
transcription factors within the vasculature more fully, we
performed morpholino knockdown of different combinations of
the four genes (Fig. 7). Translation initiation- or splice site-
targeting morpholinos were designed for each of the four genes.
The efficacy and specificity of each of these morpholinos for
their target ETS factor were verified by in vitro translation
assays (Supplemental Fig. 1). These assays showed that each
morpholino quantitatively reduced the levels of its targeted
translation product without affecting the other three genes.
Different combinations of the four oligos were injected into
single-cell embryos at three different dose levels (“low,”
Fig. 5. Expression of vascular marker genes in y11 mutants at 14 hpf
(approximately 10 somitestage). Panels show whole-mount in situ hybridization
of 14 hpf wild-type siblings (A–D) and y11 mutants (E–H) probed for flk1 (A,
E), flt4 (B, F, I), vecdn (C, G), and plxnd1 (D, H). The developing trunk region
imaged in panels A–H is noted by the box on the whole flt4-stained wild-type
embryo in panel I. Rostral is up in all panels. Scale bar=200 μm (A–H).
Fig. 4. Expression of zebrafish ETS transcription factors during somitogenesis, with flk1 and vecdn1 shown for comparison. Whole-mount in situ hybridization using
probes for zebrafish etsrp (A, E, K, Q, W), ets1 (B, F, L, R, X), fli1 (C, G, M, S, Y), fli1b (D, H, N, T, Z), flk1 (I, O, U, A′), and ve-cdn (J, P, V, B′). Embryos are
probed at the 5 somite stage (ss)/11.5 h post-fertilization (hpf) (A–D), 10 ss/14 hpf (E–J), 15 ss/16.5 hpf (K–P), and 24 h post-fertilization (Q–B′). Images in panels
A–P are complete rostral to caudal collages of multiple dorsal-view images, for a “virtual flat mount.” Images in panels Q–Vare lateral views of the entire 24 hpf
animal, while panels W–B′ are higher magnification lateral views of 24 hpf trunks. Rostral is to the left in all panels, dorsal is up in panels Q–B′. Scale bars=1000 μm
(A–V), and 250 μm (W–B′).
778 V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
“medium,” and “high” — see Materials and methods). At 24
and 48 hpf, animals were scored for the presence of trunk
circulation and the number of primary intersegmental vessel
sprouts that had emerged. These data are shown in Fig. 7. The
high morpholino doses used were titrated to just below the dose
level at which gross morphological defects and lethality began
to occur. High-dose injection of morpholinos against different
individual ETS factors led to phenotypes ranging from
relatively mild for fli1 and fli1b (partial loss of trunk circulation
at 24 hpf with full recovery of circulation in all animals by
48 hpf) to most severe for etsrp (complete loss of trunk
circulation through 48 hpf and absence of nearly all interseg-
mental vessel sprouts at 24 hpf). However, with any of the
single MO injections, 100% of intersegmental vessel sprouts
emerged by 48 hpf. The different morpholinos were also
injected in different combinations at medium (1/2 of the high
dose) and low (1/4 of the high dose) dose levels. Strong synergy
was seen between the morpholinos in the circulation and
sprouting defects they caused. When all four morpholinos were
injected together at the low dose, the resulting intersegmental
sprouting phenotype was more severe than that found for any
single morpholino at the (four times higher) high dose.
Experiments were also carried out in which morpholinos
targeting three out of the four ETS genes were injected at the
intermediate or low dose levels. In addition to confirming
synergy between ETS factors, these injections revealed a similar
ranking of the relative importance of these genes compared to
what was found in single MO injection experiments, that is,
The synergy between ETS factors can also be seen in their
effects on vascular marker expression as visualized by whole-
mount in situ hybridization (Fig. 8). Co-injection of MOs
targeting all four vascular ETS factors at “medium” dose levels
resulted in very strong reduction in transcription of either the
ETS factors themselves (Figs. 8A–D) or endothelial markers
(Figs. 8E–I). The near-complete loss of many of these markers
in animals injected with the four MOs (Figs. 8A–I) was more
pronounced than the reduced levels of expression seen in
etsrpy11null mutants (Figs. 6A–I). Furthermore, unlike etsrpy11
mutants, a strong reduction in the number of endothelial cells
was seen in 36 hpf four-morpholino injected Tg(fli1:nEGFP)y7
animals, suggesting that a significant part of the decrease in
marker expression was due to fewer expressing cells (Figs. 8L,
M). These results, together with those in Fig. 7, suggest that the
four ETS factors function combinatorially in endothelial
specification and differentiation. Interestingly, a completely
different result was observed for expression of hematopoietic
markers gata1 and gata2. Both of these genes showed little or
no reduction in their expression in animals injected with
“medium” doses of MO against all four ETS factors (Figs. 8J,
Fig. 6. Expression of ETS transcription factors and vascular and hematopoietic marker genes in y11 mutants at 24 hpf (approximately 30 somite stage). Panels show
(C), etsrp (D), flt4 (E), vecdn (F), flk1 (G), plxnd1 (H), efnb2 (I), gata1 (J), and gata2 (K). Rostral is to the left and dorsal is up in all panels. Scale bar=200 μm.
779V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
K), as compared to the reduced expression observed in etsrpy11
null mutants (Figs. 6J, K), suggesting that the hematopoietic
lineage has a more specific requirement for etsrp function.
We have taken advantage of the ease with which one can
effect combined loss-of-function on multiple members of gene
families in the zebrafish to examine the role of the ETS
transcription factor family during vascular development. Gain-
of-function studies in mice have suggested an important role for
these factors in endothelial specification and/or differentiation,
but loss-of-function studies of individual ETS factors have
generally yielded less dramatic defects in vascular development,
possibly because of extensively overlapping expression and
functional overlap between a number of different ETS factors
within the vasculature, including Ets1, Fli1, ERG, and TEL
(reviewed in Sato, 2001). Reduction in levels of the ETS-
domain protein etsrp has been reported to cause vascular
defects in zebrafish (Sumanas and Lin, 2005).
As we show in this study, there are at least three additional
ETS factors expressed within the zebrafish vasculature. We
sought to determine whether these other factors also
contribute to formation of the vasculature and what the
consequences are of combined reduction in levels of multiple
vascular ETS transcription factors. We show that the four
vascular ETS factors function in a combinatorial fashion in
the endothelium, by using morpholinos targeting etsrp and
three other vascular ETS factors (ets1, fli1, and fli1b).
Simultaneous reduction in levels of all four ETS factors by
combined morpholino knockdown leads to a near-total loss of
Morpholino injections targeting the four vascular ETS
factors show that they are not equivalent (Fig. 7), with a
relative importance etsrp>ets1>fli1≥fli1b. Reduction of etsrp
by morpholino injection causes the most severe defects in trunk
circulation and intersegmental vessel sprouting, and etsrpis able
to support some vascular differentiation on its own, in animals
injected with morpholinos against the other three factors. In
contrast, knockdown of fli1 or fli1b causes only mild vascular
Fig. 7. Trunk circulation and intersegmental vessel sprouting defects in zebrafish injected with morpholinos targeting vascular ETS factors. Morpholinos targeting fli1,
fli1b, ets1, or etsrp were injected into Tg(fli1:EGFP)y1zebrafish either alone, in combinations of three morpholinos, or all together, as noted by “x” marks at the top
and bottom of the figure. The number of animals for which phenotypes were assessed for each injection is shown at the extreme bottom of the figure (N). The upper
graphshowsthe % ofanimals withactivebloodcirculationin thetrunkat 24hpf (purple bars)and48hpf (redbars). Themiddlegraphshowsthe % ofanimals at24 hpf
with 1–15 (orange bars) or >15 (blue bars) intersegmental vessel sprouts in the trunk proper, as visualized by microscopic examination of fli1:EGFP transgene
expression in developing blood vessels. The lower graph shows the % of animals at 48 hpf with 1–15 (orange bars) or >15 (blue bars) intersegmental vessel sprouts in
the trunk proper. The graphs also include tabulation of circulation and intersegmental vessel sprout phenotypes measured in control morpholino injected animals, all of
which had trunk circulation and a full complement of trunk intersegmental vessel sprouts.
780V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
defects and they have little or no capacity to support vascular
development on their own (i.e. when the other ETS factors are
knocked down). These differences could reflect differential
functional capabilities of the different ETS factors. Despite a
high degree of sequence homology, some ETS factors do bind
slightly different sites. In addition, ETS factors have been
shown to be differentially regulated by post-translational
modification and interaction with a large number of accessory
factors. Some of the differences in vascular ETS factor
requirement that we have observed could also reflect the
relative timing or amount of expression of each of the genes.
The etsrp gene is expressed strongly in both anterior and
posterior expression domains at the 5 somite stage, unlike the
other vascular ETS genes at this stage (Fig. 4). Finally, cross-
regulation of the ETS factors could also account for differences
in the relative requirement for each gene. Loss of etsrp in
etsrpy11mutants results in strong reduction in not only etsrp but
also fli1b message levels at 24 hpf (Fig. 6). Despite the
substantial quantitative differences in relative importance of the
four ETS factors, there do not appear to be obvious qualitative
differences in their morpholino vascular phenotypes, and for the
strongest vascular phenotype reduction in multiple factors is
required. In contrast, our data suggest that etsrp plays a more
critical role in maintenance of hematopoietic development.
Although gata1 and gata2 expression are normal in etsrpy11
mutants during early somitogenesis (14 hpf/10 somites, data not
shown), nearly complete loss of the expression of gata1 and
strong reduction in gata2 are observed at 24 hpf (Fig. 6). In
contrast, injection of morpholinos against all four vascular ETS
factors at “medium” dose levels results in little or no loss of
expression of these genes despite dramatic effects on vascular
markers (Fig. 8). This presumably reflects residual etsrp protein
present in animals injected with “medium” doses of the etsrp
From our molecular characterization, it is clear that the
etsrpy11mutation represents a null mutant for etsrp, with an
early stop codon predicted to terminate the translated peptide
early in the protein, well before the essential ETS DNA binding
domain. Morpholino knockdown results in a phenocopy of the
mutant defect. An earlier report of etsrp morpholino knockdown
described a somewhat more severe vascular phenotype, with
loss of nearly all endothelial cells (as measured by flk1-GFP
transgene expression) at the highest morpholino dose levels
(Sumanas and Lin, 2005). The reasons for this difference are not
clear, but it could partly reflect different vascular-specific
transgenic lines used to visualize vessels (fli-EGFP in our study
Fig. 8. Reduction of all four vascular ETS transcription factors by morpholino knockdown. (A–K) Expression of ETS transcription factors and vascular and
hematopoietic marker genes in animals injected with either a control morpholino (top of each panel) or a cocktail of four morpholinos targeting the fli1, fli1b, ets1, and
etsrp genes (bottom of each panel). The four morpholino cocktail was injected at the “medium” dose, and an equivalent dosage of morpholino (ng) was injected in
control animals. Panels show whole-mount in situ hybridization of the trunks of 24 hpf morpholino-injected animals probed for zebrafish fli1 (A), fli1b (B), ets1 (C),
etsrp (D), flt4 (E), vecdn (F), flk1 (G), plxnd1 (H), efnb2 (I), gata1 (J), and gata2 (K). (L, M) Confocal images of the mid-trunk of 36 hpf control (L)- and four
morpholino cocktail (M)-injected Tg(fli1:nEGFP)y7animals, collected using identical microscope settings. (L) Control animals display numerous brightly EGFP-
positive endothelial nuclei in both axial (arrowheads) and intersegmental (arrows) vessels. (M) Only a small number of residual moderately EGFP-positive cells are
seen in the position of the axial vessel (arrows) in four morpholino-injected animals. Anterior is to the left in all images. Scale bar=250 μm.
781V.N. Pham et al. / Developmental Biology 303 (2007) 772–783
vs. flk-GFP in Sumanas et al.), differences in the developmental
stages at which marker genes were assayed, or toxicity of high
doses of injected morpholinos. In our hands, as noted above, a
more complete defect in vascular development requires
combined morpholino targeting of multiple ETS factors.
In conclusion, we have shown that ETS factors function
combinatorially in the zebrafish vasculature and that the
function of these factors is essential for endothelial specification
and differentiation. These factors do not act equivalently,
however, since the functional requirement for some of them is
much greater than for others. Additional work will need to be
done to further explore the differences in functional requirement
for the different ETS factors and determine whether any of these
differences reflect differing functional capabilities of the
The authors would like to thank the members of the
Weinstein laboratory for discussions and technical assistance.
We also thank Dr. Igor B. Dawid for critical reading of the
manuscript. NDL was supported by an NSF fellowship. This
research was supported (in part) by the Intramural Research
Program of the NIH.
Appendix A. Supplementary data
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