2004 Nature Publishing Group http://www.nature.

com/naturebiotechnology

LETTERS

Chemical suppression of a genetic mutation in a

zebrafish model of aortic coarctation
Randall T Peterson1, Stanley Y Shaw1,2, Travis A Peterson1, David J Milan1, Tao P Zhong1,3, Stuart L Schreiber2,
Calum A MacRae1 & Mark C Fishman1,4
Conventional drug discovery approaches require a priori
selection of an appropriate molecular target, but it is often
not obvious which biological pathways must be targeted to
reverse a disease phenotype1,2. Phenotype-based screens offer
the potential to identify pathways and potential therapies that
influence disease processes. The zebrafish mutation gridlock
(grl, affecting the gene hey2) disrupts aortic blood flow in a
region and physiological manner akin to aortic coarctation in
humans35. Here we use a whole-organism, phenotype-based,
small-molecule screen to discover a class of compounds
that suppress the coarctation phenotype and permit survival
to adulthood. These compounds function during the
specification and migration of angioblasts. They act to
upregulate expression of vascular endothelial growth factor
(VEGF), and the activation of the VEGF pathway is sufficient
to suppress the gridlock phenotype. Thus, organism-based
screens allow the discovery of small molecules that ameliorate
complex dysmorphic syndromes even without targeting the
affected gene directly.
Genetic suppressor screens may identify second-site mutations that
modify the effect of an existing genetic mutation. By screening for
phenotypic rescue, this approach identifies components of pathways
and parallel pathways otherwise difficult to identify by biochemical
means6. We reasoned that screens for chemical suppressors of a genetic
mutation, carried out in a vertebrate model organism, might provide
insights into vertebrate-specific processes, permit temporal dissection
of their timing and provide small-molecule drug-like leads.
To test the feasibility of this approach, we screened for chemical suppressors of the zebrafish gridlock (grl) mutation, which affects the
gene hey2. Zebrafish hey2 is an ortholog of the mammalian hey2 gene
(also known as HRT2, CHF1, HERP1, HESR2) and encodes a bHLH
transcriptional repressor3. Zebrafish grl m145/m145 mutants, which have
hypomorphic mutations in the hey2 gene, suffer from a malformed
aorta that prevents circulation to the trunk and tail3,4. The embryos
develop collateral vessels around the blockage that are sometimes sufficient to permit survival. Both the location of the aortic deformities

and the compensatory collaterals are similar to congenital dysplasias of

the aorta in humans such as coarctation of the aorta5. In the mouse,
null mutations of the hey2 gene cause deformities of the major vessels
and also defects of the heart79.
We arrayed grl m145/m145 embryos in 96-well plates, exposed them to
small molecules from a structurally diverse chemical library and
examined their circulation after 48 h of development. Two of 5,000
small molecules examined suppressed the gridlock phenotype in
grl m145/m145 embryos (Fig. 1), restoring circulation to the tail (Fig. 1b).
Restoration of tail circulation did not involve changes in the number
of collaterals or the timing of their formation. Instead, the morphological defect in the aorta itself was resolved, and normal flow through the
aorta occurred, as confirmed by microangiography (Fig. 1a,b) and histology (Fig. 1e,f).
The two suppressors identified are structurally related compounds
(Fig. 1c) that rescue the gridlock phenotype in a dose-dependent manner (Fig. 1d). The compound GS4012 is a more potent suppressor than
GS3999, and so it was used for all subsequent experiments. Treatment
with 2.5 g/ml GS4012 rescues circulation to the tail in approximately
55% of grl m145/m145 embryos (167 of 301 embryos tested), whereas
treatment with 7.5 g/ml GS4012 rescues circulation to the tail in all
grl m145/m145 embryos (46 of 46 embryos tested, see Fig. 1d). Other than
the correction of the aortic defect, no changes in vascular morphology
were apparent when embryos were treated with GS4012 at these concentrations, although some vascular abnormalities were observed at
higher doses.
Angioblasts in zebrafish are first evident around 14 h postfertilization (h.p.f.), and by 16 h.p.f. they begin to aggregate at the midline10,11.
The lumen of the aorta begins to form in the posterior trunk at
17.5 h.p.f., but is not complete until just before the initiation of circulation 24 h.p.f. Similarly, the critical period for GS4012 suppression
was between 12 and 24 h.p.f. (Fig. 2a). Little or no suppression was
observed when treatment began after 24 h.p.f. When GS4012 was
added during gastrulation and washed away at later and later time
points, the effectiveness of suppression was increased, reaching
maximal suppression by 30 h.p.f. Suppression was maintained after
removal of the suppressor, and rescued mutants remained viable

1Developmental Biology Laboratory, Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA and Department of
Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA. 2Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology, Harvard
University, Cambridge, Massachusetts 02138, USA. 3Present address: Department of Medicine, Vanderbilt School of Medicine, Nashville, Tennessee 37232, USA.
4Present address: Novartis Institutes for Biomedical Research, Cambridge, Massachusetts 02139, USA. Correspondence should be addressed to R.T.P.
(peterson@cvrc.mgh.harvard.edu).

Published online 18 April 2004; doi:10.1038/nbt963

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LETTERS
c

d
S
N

O
CH 3
S

O2N

100

Tail circulation (%)

80
60
40
20
0
2.5

7.5

10

Concentration (g/ml)

b
e

f
A
V

A
V

Figure 1 Chemical rescue of a genetic cardiovascular defect. (a) Microangiogram of an untreated grl m145/m145 embryo 48 h.p.f. (b) Microangiogram of a
grl m145/m145 embryo 48 h.p.f. treated with the small molecule GS4012 beginning 6 h.p.f. (c) Chemical structures of the two small molecules identified as
gridlock suppressors by whole-organism chemical screening. Top, GS4012, 4-[2-(4-methoxy-phenylsulfanyl)-ethyl]-pyridine. Bottom, GS3999, 4-[2-(4-nitrophenylsulfanyl)-ethyl]-pyridine. (d) Dose response curve for suppression of the gridlock mutant phenotype by GS4012. grlm145/m145 embryos were exposed
to the indicated concentrations of GS4012 beginning 6 h.p.f. and were transferred to embryo buffer without GS4012 at 30 h.p.f. Embryos were scored for
a circulation to the tail 48 h.p.f. Tail circulation percent represents the number of embryos with tail circulation divided by total embryos with circulation
anywhere (e.g., cranial circulation). (ef) Transverse sections comparing the histology of the aorta in grlm145/m145 embryos left untreated (e) or treated with
7.5 g/ml GS4012 (f). A, dorsal aorta; V, cardinal vein.

We used real-time PCR to quantify the expression of several genes

believed to play roles in angioblast cell fate determination or migration. Expression of one of these genes, VEGF, was upregulated
in grl m145/m145 embryos by exposure to GS4012 (Fig. 2b). Treatment
with GS4012 also increased VEGF mRNA levels in wild-type embryos

Rescue (%)

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50

P < 0.002

60
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(relative units)

Percentage change

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P < 0.025

wt

grl wt5 wt7.5

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shh

flt4

grl efnB2 vegf

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Percentage change

to adulthood. Therefore, suppression is irreversible and largely

complete before actual formation of the aorta. Furthermore, the
suppressors activity is required precisely during the period of
angioblast cell fate determination and migration, suggesting a direct
role in these processes.

VEGF induction (fold)

2004 Nature Publishing Group http://www.nature.com/naturebiotechnology

50

Figure 2 Chemical suppressors of gridlock act before

aorta formation and upregulate VEGF expression.
(a) Timing of gridlock suppression by GS4012.
grl m145/m145 embryos were treated with 2.5 g/ml
GS4012 at the indicated times and allowed to develop
to 48 h.p.f. (triangles) or treated with 7.5 g/ml GS4012
at 5 h.p.f. and allowed to develop until the indicated
times, at which point they were transferred to embryo
buffer without GS4012 and allowed to develop to 48
h.p.f. (squares). The rescue percentage for each time
point represents the difference between the percentages
of wild-type circulation observed in treated and untreated
embryos. (b) The effect of GS4012 on expression of
genes involved in formation of the aorta. Asterisk, not
significant. (c) The effect of GS4012 treatment on VEGF
mRNA levels as determined by quantitative RT-PCR.
Percent change relative to wild-type DMSO-treated
embryos (wt) is shown for grl m145/m145 embryos (grl)
and wild-type embryos treated with 5 g/ml (wt5) or
7.5 g/ml (wt7.5) GS4012. (d) Expression of hey2
(diamonds) and VEGF (squares) mRNA during
development. Message quantity was normalized to
glyceraldehyde 3-phosphate dehydrogenase quantity at
each time point and is expressed in relative units, with
the most abundant time point quantity arbitrarily set
at 10. (e) Time course of VEGF induction by GS4012.
Fold induction represents the ratio of VEGF mRNA in
treated versus untreated embryos. Error bars indicate
the standard deviation in three replicates.

VOLUME 22 NUMBER 5 MAY 2004 NATURE BIOTECHNOLOGY

(Fig. 2c). VEGF plays an important role in formation of the aorta.

VEGF122 expression in the Xenopus hypochord acts as a chemoattractant, stimulating angioblast migration to the midline before aorta formation12. Targeted disruption of VEGF in mice causes severely
disrupted aortic development13,14, and a VEGF promoter haplotype is
associated with an increased risk for cardiovascular defects (including
coarctation of the aorta) in humans with deletions in the DiGeorge
locus 22q11 (ref. 15). In zebrafish, hey2 and VEGF transcript levels are
temporally correlated throughout development (Fig. 2d). Both genes
are upregulated between 10 and 24 h.p.f., the period of greatest sensitivity to gridlock suppression, and are expressed in adjacent tissues
during this period3,16. VEGF induction by GS4012 treatment is
observed at many stages of development (Fig. 2e).
To test whether the increased VEGF expression induced by GS4012
could be responsible for suppression of the gridlock phenotype, we
injected grl m145/m145 embryos with an expression plasmid containing
the murine VEGF cDNA (pmVEGF). Approximately 5% of uninjected
or vector-injected embryos showed wild-type circulation. (The penetrance of the gridlock phenotype is typically 95% but varies slightly
from clutch to clutch, possibly reflecting the existence of unidentified
modifier genes). However, about one-third of the mutant embryos
injected with pmVEGF showed wild-type circulation to trunk and tail
(Fig. 3). Therefore, VEGF overexpression is sufficient to suppress the
mutant phenotype in grl m145/m145 embryos. Chemical suppression of
the gridlock phenotype appears to be more efficient than injection of
pmVEGF. This may be because plasmid-driven VEGF expression is
mosaic or because the chemical suppressors have additional mechanisms of action beyond VEGF induction.
Because the gridlock suppressors promote vessel formation in
zebrafish, we wondered if they would have similar activity in a mammalian system. We tested the effect of GS4012 on endothelial cell
tubule formation using cultured human cells. Human umbilical vein
endothelial cells (HUVECs) treated with GS4012 formed tubule networks that appeared more extensive and robust than those formed by
untreated HUVECs (Fig. 4a,b) and were comparable to those formed
by HUVECs treated with recombinant VEGF (Fig. 4c,d). Tubules
formed in the presence of GS4012 covered nearly twice as much
matrix surface area as untreated tubules (Fig. 4d). Therefore, the gridlock suppressor appears to promote vessel formation in mammalian as
well as zebrafish systems. Given that GS4012 promotes VEGF expression in zebrafish, we reasoned that GS4012 might promote tubule formation in human cells through increased VEGF expression. We used
quantitative PCR to test VEGF transcript levels in HUVEC cells. Cells
treated with GS4012 express significantly (P = 0.04) more VEGF than
cells treated with dimethyl sulfoxide (DMSO) (Fig. 4e), suggesting that
the increased VEGF expression in HUVECs treated with GS4012 may
contribute to the increased tubule formation in those cells.
The hey2 gene encodes a transcriptional repressor that drives
angioblast differentiation to an arterial fate rather than a venous fate17.
It appears to function downstream of Notch in this bimodal cell fate
decision1719. VEGF plays several roles in vessel formation, including
stimulating arterial differentiation, expanding the number of
angioblasts and drawing them towards the midline during vessel formation12,20,21. Thus, it is possible that rescue of the gridlock mutation
by VEGF-inducing GS4012 treatment or by VEGF cDNA injection
reflects stimulation of these compensatory processes. Regardless of the
exact nature of the relationship between hey2 and VEGF, it is clear that
they are related functionallyoverexpression of one compensates for
hypomorphic mutation of the other. These data are consistent with
genetic studies that place hey2 downstream of VEGF and Notch signaling in arterial differentiation pathways20. Although GS4012 induces

NATURE BIOTECHNOLOGY VOLUME 22 NUMBER 5 MAY 2004

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Circulation to the tail (%)

2004 Nature Publishing Group http://www.nature.com/naturebiotechnology

LETTERS
74/231

30
25
20
15
10
5

19/418

9/187

NI

pCS2

0
VEGF

Figure 3 VEGF cDNA injection suppresses the gridlock mutation. (a) The
percentage of grl m145/m145 embryos showing a circulation to the tail that
are not injected (NI), injected with empty plasmid (pCS2) or with plasmid
containing the murine VEGF cDNA. Circulation was assessed 48 h.p.f.,
and results are expressed as the number of embryos with a circulation to
the tail divided by the number of embryos with a circulation to the head.
(b,c) Microangiograms of grl m145/m145 embryos not injected (b) or injected
with pmVEGF (c).

expression of VEGF, and VEGF overexpression is sufficient to suppress

the gridlock phenotype, it remains possible that VEGF induction is not
the primary mechanism by which GS4012 suppresses the gridlock
phenotype in vivo. For example, in addition to its effects on VEGF
expression, GS4012 may also stimulate compensatory pathways that
are parallel to or downstream of VEGF signaling.
Beyond their utility for developmental biological studies, zebrafish
appear to be well suited for modeling genetic diseases. Several mutations identified by forward genetic screening cause phenotypes in
zebrafish that are similar to human congenital disease phenotypes22,23.
In a number of cases where the mutations in human and zebrafish
have been identified, orthologous genes are responsible for the human
and zebrafish phenotypes24,25. Furthermore, the physiological effects
of many drugs have been shown to be comparable in humans and
zebrafish26,27. Therefore, insights gained from zebrafish suppressor
screens may be directly applicable to human disease. One shortcoming
of conventional approaches to developing therapies for genetic diseases has been the difficulty of determining what molecular targets
could compensate for the effects of the mutated genes. This study
demonstrates the feasibility of unbiased screening for such targets
directly in a whole, vertebrate organism.

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LETTERS
c

e
120,000
100,000
80,000
60,000
40,000
20,000
0
DMSO GS4012 VEGF

Relative VEGF expression

d
Tubule formation
(arbitrary units)

2004 Nature Publishing Group http://www.nature.com/naturebiotechnology

P = 0.04
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
NT 2 h 4 h

Figure 4 GS4012 promotes endothelial cell tubule formation. (ac) Networks of tubules formed by HUVECs treated with DMSO (a), 5 g/ml
GS4012 (b) or 10 ng/ml recombinant VEGF (c). (d) The extent of tubule
formation in HUVECs treated with DMSO, GS4012 or VEGF. Tubule
formation was quantified as described in Methods. Error bars represent
the standard deviation. (e) Relative VEGF expression levels in HUVECs
treated with DMSO (NT) or with 5 g/ml GS4012 for 2 h or 4 h. Message
quantity for each sample was normalized to glyceraldehyde 3-phosphate
dehydrogenase quantity and is expressed in relative units, with the DMSOtreated value arbitrarily set at 1.

Gene therapy has been viewed as a potential means of compensating

for genetic mutations, but several obstacles have limited the success of
this approach28. For example, gene transfer to the recipient is inefficient and not uniform, and once the gene is transferred, it remains difficult to control the level of expression of that gene. Concerns have also
been raised about the safety of the viral vectors used in gene therapy29.
A small moleculebased approach offers a number of advantages.
Small molecules can be administered more efficiently and uniformly
without the use of viral vectors. Most important, dosing can be controlled throughout the process, making it possible to maintain an
effect for long periods of time or to terminate exposure as needed.
Recently, it has become possible to isolate zebrafish lines with mutations in virtually any gene30. By combining this technology with the
suppressor screening approach described here, it should be possible to
model many disease processes and identify small molecules capable of
modifying those processes in new ways.
METHODS
Zebrafish care. All zebrafish experiments were approved by the Massachusetts
General Hospital Subcommittee on Research Animal Care.
Screening for suppressors of the gridlock mutation. Forty pairs of homozygous grl m145/m145 zebrafish were mated, and fertilized eggs were arrayed three
embryos per well in 96-well plates containing 0.25 ml E3 embryo buffer (5 mM
NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). At the 50% epiboly
stage, small molecules from the DiverSet E library (Chembridge) were added to
each well, yielding a final concentration of approximately 2 g/ml. Embryos
were incubated at 28.5 C for 48 h. Circulation to the tail was assessed visually
using a dissecting microscope.
Quantitative PCR. Total RNA was extracted from groups of embryos using the
TRIZOL method and reverse transcribed using random priming and

598

SuperScript II following the manufacturers instructions. Five groups of

20 grl m145/m145 embryos were left untreated or treated with 7.5 g/ml GS4012 at
6 h.p.f. (Fig. 2b). Expression was quantified at 24 h.p.f. and is depicted as the
percentage change in transcript levels in GS4012-treated embryos relative to
transcript levels in control-treated embryos. The P value represents significance
in the pairwise comparison of transcript levels between GS4012-treated and
control embryos as determined using the paired t-test. For Figure 2c, four
groups of 40 embryos were used for each condition. VEGF levels were normalized in relation to -actin expression. P values represent significance as determined using the paired t-test. In Figure 2e, grl m145/m145 embryos were treated
with 7.5 g/ml GS4012 at 6 h.p.f. and allowed to develop for the indicated
length of time. mRNA levels were normalized to -actin expression. For all
experiments, cDNA was quantified using an Applied Biosystems Sequence
Detection System 7000. Taqman probes were used for hey2, VEGF, EfnB2a and
glyceraldehyde 3-phosphate dehydrogenase (G3PDH) cDNAs. The SYBR green
method was used to quantify sonic hedgehog and Flt4 cDNAs. Primer and
probe sequences used were as follows: hey2, 5-GTCCGACAGCGACATGGAT3, 5-ACCATTGCTTTGGCCAGAGT-3, probe 5-6FAM-AAACCATTGATG
TGGGCAGCCAGAATAA-MGBNFQ-3; zebrafish VEGF, 5-TGCTCCTGCAA
ATTCACACAA-3, 5-ATCTTGGCTTTTCACATCTGCAA-3, probe 5-6FAMTGCAATGCAAGTCCAGACA-MGBNFQ-3; human VEGF, 5-CCAAGGCC
AGCACATAGGA-3, 5-CTTTCTTTGGTCTGCATTCACATTT-3, probe 56FAM-ATGAGCTTCCTACAGCAC-MGBNFQ-3; EfnB2a, 5-GGAACACCA
CGAACACCAAGT-3, 5-TCCCCAATCTGCGGATACAG-3, probe 5-6FAMTGCCGGGACAGGGTCTGGT-MGBNFQ-3; zebrafish G3PDH, 5-GATAAC
TTTGTCATCGTTGAAGGTCTT-3, 5-CGGTCTTCTGTGTTGCTGTGA-3,
probe 5-VIC-TGAGCACTGTTCATGC-MGBNFQ-3; human G3PDH, assayson-demand reagents (Applied Biosystems); sonic hedgehog, 5-ACAATCCCG
ACATTATCTTTAAGGA-3, 5-TGTCTTTGCATCTCTGTGTCATGA-3; Flt4,
5-CTGCTCTGGGAGATTTTCTCACTAG-3, 5-TCGTTTGCAGAAATCTTC
ATCAA-3.
cDNA injections. Embryos generated by mating homozygous grl m145 mutants
were injected at the 1- to 4-cell stage with 1 nl pCS2+ vector or pCS2+-containing murine VEGF cDNA at a concentration of 20 ng/l in 0.3 Danieaus buffer
(17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2 and 1.5 mM
HEPES, pH 7.6). Embryos were incubated at 28.5 C for 48 h. Circulation to the
tail was assessed visually using a dissecting microscope.
Fluorescence microangiography. Fluorescence microangiography was done as
described4.
Endothelial cell tubule formation assay. Passage 4 HUVECs maintained in
EGM-2-MV BulletKit medium (Cambrex) were treated for 11 h with GS4012
(5 g/ml), VEGF (10 ng/ml; R&D Systems) or DMSO dissolved in medium.
Cells were trypsinized, resuspended in M199 medium containing 1% FBS
(GIBCO-Invitrogen) and either GS4012, VEGF or DMSO at the same concentrations as before and plated into a 48-well plate precoated with 100 l of
Growth Factor Reduced Matrigel (BD Biosciences). After 14 h at 37 C and
5% CO2, wells were carefully washed once with PBS and photographed
under low magnification. The extent of tubule formation was quantified by
counting the number of pixels whose signal intensity exceeded a defined
threshold (MetaMorph software, Universal Imaging). Image processing and
analysis were standardized for all samples and experiments were done in triplicate or quadruplicate.
ACKNOWLEDGMENTS
This work was supported by grants from the National Institutes of Health, by
the Ned Sahin Research Fund for Restoring Developmental Plasticity and by a
sponsored research agreement with Novartis Institutes for Biomedical Research.
S.Y.S. is a Physician-Postdoctoral Fellow and S.L.S. is an Investigator at the
Howard Hughes Medical Institute. S.L.S. is also supported by a grant from the
Donald W. Reynolds Cardiovascular Clinical Research Center (University of Texas
Southwestern Medical Center).
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests (see the Nature Biotechnology
website for details).
Received 27 October 2003; accepted 13 February 2004

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Published online at http://www.nature.com/naturebiotechnology/

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