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Supplementary Information:
Supplementary Fig. 1: EGFL7 is conserved during vertebrate evolution.
a: Amino acid alignment among human, mouse, xenopus and zebrafish EGFL7s. The Egfl7 gene encodes a
putative secreted protein of ~30kD. The human (Homo sapiens) amino acid sequence shares 77.45 %,
47.14 % and 42.96 % homology to that of the mouse (Mus musculus), frog (Xenopus laevis) and zebrafish
(Danio rerio), respectively. Structural analysis using a number of algorithms predicts that the EGFL7
proteins contain the following domains: a signal sequence (black box), an EMI domain at the N-terminus1
(blue box), two EGF-like domains in the central portion (green boxes), followed by a leucine and valine
rich C-terminal region.
b: Zebrafish Egfl7 cDNA, amino acid, and an intron sequence (red). Arrowed lines indicate the two
antisense oligos, AS-47 and AS195, and the PCR primers used to detect intron retention. AS-47 hybridizes to
the 5’ UTR and was expected to block translation. AS195 targets the splicing of the intron labeled "Targeted
intron". It is expected to cause retention of this intron, and hence premature translation termination of
EGFL7 at amino acid 69. Red triangle = an intron upstream of the targeted exon. Randomized controls for
the two antisense oligos are:
Con-47 =5'-ACGACGGTCACGATGAATGGAGAGT-3'
Con195=5'-CATTGTTCATCGTCTTGTTGCGTGT-3'.
c: Homology comparison among EGFL7 and EGFL8 from different species. We searched the human and
mouse genomic databases and identified only one closely related gene Egfl8 (EGF-like domain 8) / NG3
(New Gene 3). The mammalian EGFL7 and mammalian EGFL8 have identical domain organization (data
not shown). Interestingly, thus far we have not been able to identify an EGFL8 orthologue in several fish
genomes (Danio rerio, Medaka and Fugu). Therefore, we hypothesize that in lower vertebrates, there is
only one member in this gene family. Although the zebrafish EGFL7 also shares some homology to the
mammalian EGFL8, we believe that it is a true orthologue of the mammalian EGFL7 for the following
reasons: first, the zebrafish EGFL7 amino acid sequence is more homologous to the mammalian EGFL7
than to EGFL8 as shown here; second, the zebrafish Egfl7 locus is syntenic to the human Egfl7 locus; third,
the fish and mammalian Egfl7 expression patterns are strikingly similar. The zebrafish and mammalian
Egfl7 are each expressed in the vascular system and absent from the nervous system (see main text),
whereas the mammalian Egfl8 is expressed in a subset of blood vessels as well as in the peripheral nervous
system (data not shown).
Method for sequence identification: Human and mouse EGFL7 sequences were identified from public
databases by searching for proteins with a signal peptide or contain domains that are frequently found in
secreted proteins or receptors 2. Xenopus Egfl7 is a rough draft derived from a single genbank entry
BC044267. Zebrafish Egfl7 was cloned by low stringency PCR and subsequent screening of a cDNA
library made from 24 hpf embryos. EGFL8 was identified by BLAST using EGFL7 sequences.
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Radiation hybrid mapping experiments using the T51 panel place the zebrafish Egfl7 in linkage group 21
(LG21), close to the EST marker fk20d12.y1 (Accession number AW566846). This region of zebrafish
LG21 appears to be syntenic with human chromosome 9q33 to 9q34, the locus where human Egfl7 resides.
The following genes are found within this human locus: Notch1 (9q34), carboxyl ester lipase (CELL;
9q34), and proteasome subunit beta 7 (psmb7; 9q33). These genes are present in the region of LG21 where
zebrafish Egfl7 mapped (LG21, 19.6-29.0 CM).
Supplementary Fig. 2: Human and murine Egfl7 is expressed in vasculatures associated with active cell
proliferation, and is down regulated in mature vasculatures.
Egfl7 radioactive in situ hybridization in human (b, d, f) and mouse (h, j) tissue sections, and the
corresponding H&E staining (a, c, e, g, i). a and b: Body cavity of a 14 week old human fetus showing
expression in the endothelia of arteries (A), veins (V) and capillaries (C). k: Immuno-fluorescent staining
of an adult mouse lung section with endoglin (green) and EGFL7 (red). Yellow staining indicates vessels
that are positive for both Endoglin and EGFL7. Only a small subset of vessels expresses EGFL7 (white
arrows), note that many large vessels and almost all capillaries do not express EGFL7.
The mouse Egfl7 transcript is first detected at E7.0 in embryonic vascular primordia such as the allantois,
which mainly consists of endothelial progenitors3,4. Egfl7 continues to be expressed by ECs in all vessel
types (artery, vein and capillary) throughout embryonic and early postnatal development (data not shown).
Egfl7 expression is down regulated in most adult organs (j and data not shown) such that the transcripts are
detectable only in a few highly vascularized organs including the lung, kidney and heart. Even in these
highly vascularized tissues, only a subset of vessels has detectable level of the EGFL7 protein (k). Egfl7 is
strongly up-regulated in proliferating tissues such as the deciduas in the pregnant mouse uterus; tumors
from the mmtv-Her2 and mmtv-Wnt1 transgenic models; and numerous xenograft tumor tissues (data not
shown).
Radioactive in situ hybridization of human Egfl7 was carried out on multiple samples of the following adult
tissues: lung (Fig. 1e, 1h), kidney, heart, stomach, small intestine, colon, spleen, liver, pancreas, brain,
bone, testis, prostate and muscle, all showed low to undetectable expression (data not shown). Supporting
the notion that Egfl7 is up-regulated in actively growing / remodeling blood vessels, we found strong
expression in the vascular endothelia of many human tumors, including: lung adenocarcinoma (Fig. 1f, 1i),
clear cell renal cell carcinoma (e, f), prostate and ovarian carcinoma, gastric carcinoma, osteosarcoma, and
chondrosarcoma (data not shown). Furthermore, Egfl7 mRNA is barely detectable in the nearby non-tumor
vasculature from the same patients (data not shown).
Methods: Radioactive in situ hybridization: tissues were processed for in situ hybridization by a method
described previously 5. 33P-UTP-labelled RNA probes were generated as described 6. Egfl7 sense and
antisense probes were synthesized from two human cDNAs and one murine cDNA that correspond to
nucleotides 382 to 1062, –137 to 150, and 173 to 774, respectively. (Nucleotide A in the initiation codon is
counted as nucleotide #1). Wholemount in situ hybridizations were carried out as described 7 with slight
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modifications. Stained embryos were cleared in a 2:1 mixture of benzyl alcohol/benzyl benzoate for
photography. Sense and antisense riboprobes were made using the following templates: a murine Egfl7
partial cDNA clone (IMAGE clone 519249, Genbank accession: AA107358); a zebrafish Egfl7 partial
cDNA clone corresponding to nucleotides 169 to 807; all other molecular markers were from the Fishman
(MGH), Stainier (UCSF) and Weinstein (NIH) labs. Following whole-mount in situ hybridization analysis,
embryos were re-fixed in 4 % paraformaldehyde (PFA) in 1XPBS, dehydrated through an ethanol series,
and embedded in JB-4Plus plastic (Polysciences). 5 m sections were counterstained with 0.2 % nuclear
fast red, and mounted in Cytomount 60.
Supplementary Fig. 3: Egfl7 knockdown causes specific vascular tube formation defect.
a: Quantitative phenotypic evaluation of Egfl7 knockdown embryos (AS-47, red) vs. control (Con-47, blue)
embryos at 30 hpf. 40 % of 516 antisense oligo injected embryos displayed overt vascular defects such as
no circulation or having an incomplete circulatory loop. Only 3 % of 390 control oligo injected embryos
showed mild vascular problem, such as the absence of one or two ISVs. Data are pooled from 3 separate
experiments. b: 30 hpf embryos injected with control oligo Con-47 or antisense oligo AS-47 were in situ
hybridized to flk1 (shown here), and tie1 , epherinB2, Flt4, ephB4, gridlock (data not shown). c: Dorsal
view of the head vasculatures in 30 hpf embryos injected with control oligo Con -47 or antisense oligo AS-47,
and in situ hybridized to fli1. White arrows point to the Primary Hind Brain Channels (PHBC) that clearly
have lumen in the control fish, and are lumenless in the knockdown fish. White arrowheads point to the
heart. All major vessels in the head are accounted for in the knockdown fish, but they are all lumenless. d:
Close up view of a segment of PHBC in 30 hpf flk1:GFP embryos injected with control oligo Con-47 or
antisense oligo AS-47. Lack of lumen formation is evident in the knockdown fish. e: Intron retention caused
by the antisense oligo AS195 is confirmed by RT-PCR of total RNAs isolated from six individual embryos
injected with either AS195 (lanes 1 to 3) or Con195 (lanes 4 to 6) oligos. A 382 bp band representing the
intron-retention product is seen in the AS injected fish only, whereas the 307 bp wild type band is detected
in both groups. Lanes 8 (pool of AS195 samples) and 9 (pool of Con195 samples) are no RT controls, lane 7
is DNA ladder (b.p.=base pairs). PCR strategy is summarized in the diagram on top. f: Endogenous AP
staining of 48 hpf embryos injected with Con-47 (top) or AS-47 (middle), and a 72 hpf embryo injected with
AS-47 plus Egfl7 coding RNA (bottom). Co-injection of AS-47 with Egfl7 coding RNA dampens the vascular
defects. A majority (72 %, 40/57) of the AS-47 injected embryos lack vigorous circulation, and have severe
vascular defects such as near total loss of the ISVs (middle panel). In contrast, 82 % (85/104) of the
embryos co-injected with AS-47 plus 0.5 ng Egfl7 coding RNA have functioning, though somewhat
abnormal vasculature. AP staining indicated that a significant subset of ISVs is restored
Supplementary Fig. 4: Failure of vascular tubulogenesis is a specific and primary defect in the Egfl7
knockdown embryos.
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a, b: The circulatory system of a control flk1:GFP embryo at 30 hpf is filled with orange-red labeled
microspheres (0.04 micron). GFP (a) and orange-red (b) signals are shown. c, d: Injection of orange-red
labeled microspheres fails to fill the major vessels of an age-matched Egfl7 knockdown flk1:GFP
transgenic embryo (d), the vasculature of the same fish is shown in c.
e: Primary vessel lumen formation occurs normally in the absence of blood flow. A flk1:GFP transgenic
embryo injected with the silent heart (sih) antisense oligo was fixed at 30 hpf, cross-sectioned and
counterstained with phalloidin (red) and DAPI (blue). Two distinct axial vessels (DA = aorta, PCV =
posterior cardinal vein) with lumen are evident. The sih morphants (MO) had no circulation due to the lack
of cardiac contractile function, which accurately phenocopied the sih mutant 8. Silent heart (sih) antisense
oligo = 5'-CATGTTTGCTCTGATCTGACACGCA-3'.
f: fli1 expression in 13-15 somite embryos. We found no obvious defects in the Egfl7 knockdown embryos
from around 6- to 22-somite stages, a period in which EC specification, proliferation and migration towards
the midline are actively taking place. g: The expression of fkd7, a marker for floorplate (black arrowhead);
hypochord (black arrow); gut tube (red arrow) and precordal plate mesoderm (red arrowhead), is unaltered
in the Egfl7 knockdown embryos (AS) with severe circulation defects. Interestingly, fixation and
dehydration often caused curvature of the notochord in the KDs. We speculate that the cord-like primary
vessels failed to provide adequate mechanical support for the axial structures. h,i: gata1 expression in a
control (Con) vs. a Egfl7 knockdown embryo (AS) at 15-somite stage (h) and 30 hpf (i). Gata1 expression
at 13-15 somite stage is unaffected by knocking down Egfl7. At 30 hpf, Gata1+ cells in the Egfl7
knockdown fish remain in the posterior ventral lateral region of the body where they initially developed 9.
In contrast, gata1+ cells in the control oligo injected fish are readily circulating at this time.
j-l: VEGF does not directly regulate vascular tubulogenesis. flk1:GFP transgenic fish injected with control
(j), vegf antisense oligo (k), or Egfl7antisense oligo plus a 1:1 mixture of vegf-165 and vegf-121 mRNA (l)
were analyzed at 30 hpf. Shown here are deconvolution microscopy images of mid-trunk cross sections
counter stained with DAPI (blue), these sections were taken at the level similar to what is indicated by the
white lines in Figure3e. In the most severe vegf knockdown embryos, there are fewer ECs (white arrows) in
any given section compared to the control embryos. Furthermore, some ECs show signs of apoptosis such
as DNA fragmentation (red arrow). Interestingly, the remaining healthy ECs appear to form tube-like
structures that are aberrant in shape and number. These observations are in agreement with the finding that
VEGF is an endothelial cell mitogen and survival factor 10. Our result indicates that VEGF is not directly
involved in regulating vascular tubulogenesis, therefore its function is distinct from that of EGFL7. This
conclusion is further supported by the fact that vegf mRNA fails to restore tube formation in the Egfl7
knockdown embryos (l). As expected, injection of vegf mRNA increases the number of ECs (l). The vegf
antisense oligo used in our experiment is the same as reported 11.
Con = control oligo (Con-47) injected embryos, AS = Egfl7 antisense oligo (AS-47) injected embryos, MO=
morpholino antisense oligo injected embryos.
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Supplementary Fig. 5: Egfl7 regulates de novo vascular tubulogenesis.
Serial cross sections of the trunk (a-f), or longitudinal sections of the head (g-h) were taken from the
flk1:GFP transgenic fish and stained with a tight junction marker ZO-1 (red) and GFP (green). Egfl7
knockdown (b, d, f, h) or control (a, c, e, g) fish at 22 (a-b), 24 (c-d), 26 (e-f) somite and 30 hpf (g-h)
stages were analyzed. Trunk sections were taken at the axial level indicated by white lines in Figure 3.
White arrows = arterial angioblasts, white arrowheads = venous angioblasts. Tight junctions between a pair
of arterial (red arrows) or venous (red arrowheads) angioblasts are indicated. Scale bar: 0.01mm (a, b, d, e,
f), 0.008mm (c), 0.006mm (g, h).
Supplementary Figure 6. A model depicting the key steps of vasculogenesis, using the trunk region of the
zebrafish embryo as an example.
a: In early somitogenesis stages, a bi-lateral cohort of presumptive stem / progenitor cells called
hemangioblasts (yellow) are influenced by signals (blue arrows) emanated from axial structures such as the
hypochord (H). b: These cells then commit to the hematopoietic (HSC, pink) as well as arterial (aEC, red)
and venous (vEC, blue) endothelial fates, and migrate towards the midline in response to the chemotactic
activity of midline signals (blue arrows). c: The aECs and vECs coalesce into “cord” like structure in the
midline at around 18- to 22-somite (~18-20 hpf) stages. Angioblasts form intimate connections as evident
by extensive tight junctions (red dots). d: Between 22- to 26-somite (~20-22 hpf) stages, angioblasts
undergo local migration along the dorsal-ventral (D-V) axis, and possibly along the anterior-posterior axis
as well. Cell-cell connections are refined since the massive tight junctions among angioblasts are gradually
reduced. e: By the 26-somite stage, this local migration is completed. The arterial and venous angioblasts
are now separated along the D-V axis. Connection among angioblasts are spatially defined, such that
limited point of tight junctions are found between pairs of angioblasts. At any given axial level, two to four
angioblasts of the same type (arterial or venous) align in the form of a rudimentary tube. f: Around 22-24
hpf, angioblasts undergo extensive morphological changes to form a squamous epithelium. Functional
tubes are thus established to enable circulation. g: Maturation of the nascent vessels ensues via the
recruitment of mural cells and establishment of appropriate connection with adjacent tissues. Based on the
data presented in this report, we hypothesize that during the transition from step c to step e, angioblasts
secrete EGFL7 protein (solid green lines) to create a microenvironment that is favorable for the local
movement of angioblasts, thereby enabling the subsequent assembly of major vascular tubes. EGFL7 is
down regulated or turned off in mature vessels (broken green lines). N = notochord, DA = dorsal aorta,
PCV = posterior cardinal vein.
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