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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 humans3–5. 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 heart7–9. 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 µg/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 µg/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. ([email protected]).

Published online 18 April 2004; doi:10.1038/nbt963

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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). (e–f) Transverse sections comparing the histology of the aorta in grlm145/m145 embryos left untreated (e) or treated with 7.5 µg/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

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to adulthood. Therefore, suppression is irreversible and largely complete before actual formation of the aorta. Furthermore, the suppressor’s 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

0

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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 µg/ml GS4012 at the indicated times and allowed to develop to 48 h.p.f. (triangles) or treated with 7.5 µg/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 µg/ml (wt5) or 7.5 µg/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.

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(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 decision17–19. 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 functionally—overexpression 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

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

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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. (a–c) Networks of tubules formed by HUVECs treated with DMSO (a), 5 µg/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 µg/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 molecule–based 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 µg/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

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SuperScript II following the manufacturers’ instructions. Five groups of 20 grl m145/m145 embryos were left untreated or treated with 7.5 µg/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 β-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 µg/ml GS4012 at 6 h.p.f. and allowed to develop for the indicated length of time. mRNA levels were normalized to β-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 5′6FAM-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/µl in 0.3× Danieau’s 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 µg/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 µl 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/

1. Crews, C.M. & Splittgerber, U. Chemical genetics: exploring and controlling cellular processes with chemical probes. Trends Biochem. Sci. 24, 317–320 (1999). 2. Schreiber, S.L. Target-oriented and diversity-oriented organic synthesis in drug discovery. Science 287, 1964–1969 (2000). 3. Zhong, T.P., Rosenberg, M., Mohideen, M.A., Weinstein, B. & Fishman, M.C. gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science 287, 1820–1824 (2000). 4. Weinstein, B.M., Stemple, D.L., Driever, W. & Fishman, M.C. Gridlock, a localized heritable vascular patterning defect in the zebrafish. Nat. Med. 1, 1143–1147 (1995). 5. Towbin, J.A. & McQuinn, T.C. Gridlock; a model for coarctation of the aorta? Nat. Med. 1, 1141–1142 (1995). 6. St. Johnston, D. The art and design of genetic screens: Drosophila melanogaster. Nat. Rev. Genet. 3, 176–188 (2002). 7. Sakata, Y. et al. Ventricular septal defect and cardiomyopathy in mice lacking the transcription factor CHF1/Hey2. Proc. Natl. Acad. Sci. USA 99, 16197–16202 (2002). 8. Donovan, J., Kordylewska, A., Jan, Y.N. & Utset, M.F. Tetralogy of fallot and other congenital heart defects in hey2 mutant mice. Curr. Biol. 12, 1605–1610 (2002). 9. Gessler, M. et al. Mouse gridlock: no aortic coarctation or deficiency, but fatal cardiac defects in hey2–/– mice. Curr. Biol. 183, 37–48 (1997). 10. Eriksson, J. & Lofberg, J. Development of the hypochord and dorsal aorta in the zebrafish embryo (Danio rerio). J. Morphol. 244, 167–176 (2000). 11. Fouquet, B., Weinstein, B.M., Serluca, F.C. & Fishman, M.C. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Dev. Biol. 183, 37–48 (1997). 12. Cleaver, O. & Krieg, P.A. VEGF mediates angioblast migration during development of the dorsal aorta in Xenopus. Development 125, 3905–3914 (1998). 13. Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435–439 (1996). 14. Ferrara, N. et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439–442 (1996). 15. Stalmans, I. et al. VEGF: a modifier of the del22q11 (DiGeorge) syndrome? Nat. Med.

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9, 173–182 (2003). 16. Liang, D. et al. The role of vascular endothelial growth factor (VEGF) in vasculogenesis, angiogenesis, and hematopoiesis in zebrafish development. Mech. Dev. 108, 29–43 (2001). 17. Zhong, T.P., Childs, S., Leu, J.P. & Fishman, M.C. Gridlock signaling pathway fashions the first embryonic artery. Nature 414, 216–220 (2001). 18. Iso, T., Chung, G., Hamamori, Y. & Kedes, L. HERP1 is a cell type-specific primary target of Notch J. Biol. Chem. 277, 6598–6607 (2002). 19. Nakagawa, O. et al. Members of the HRT family of basic helix-loop-helix proteins act as transcriptional repressors downstream of Notch signaling. Proc. Natl. Acad. Sci. USA 97, 13655–13660 (2000). 20. Lawson, N.D., Vogel, A.M. & Weinstein, B.M. Sonic hedgehog and vascular endothelial growth factor act upstream of the Notch pathway during arterial endothelial differentiation. Dev. Cell 3, 127–136 (2002). 21. Ash, J.D. & Overbeek, P.A. Lens-specific VEGF-A expression induces angioblast migration and proliferation and stimulates angiogenic remodeling. Dev. Biol. 223, 383–398 (2000). 22. Shin, J.T. & Fishman, M.C. From Zebrafish to human: modular medical models. Annu. Rev. Genomics Hum. Genet. 3, 311–340 (2002). 23. Dooley, K. & Zon, L.I. Zebrafish: a model system for the study of human disease. Curr. Opin. Genet. Dev. 10, 252–256 (2000). 24. Xu, X. et al. Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin. Nat. Genet. 30, 205–209 (2002). 25. Roman, B.L. et al. Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development 129, 3009–3019 (2002). 26. Langheinrich, U. Zebrafish: a new model on the pharmaceutical catwalk. Bioessays 25, 904–912 (2003). 27. Milan, D.J., Peterson, T.A., Ruskin, J.N., Peterson, R.T. & MacRae, C.A. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish. Circulation 107, 1355–1358 (2003). 28. Thomas, C.E., Ehrhardt, A. & Kay, M.A. Progress and problems with the use of viral vectors for gene therapy. Nat. Rev. Genet. 4, 346–358 (2003). 29. Check, E. Cancer risk prompts US to curb gene therapy. Nature 422, 7 (2003). 30. Wienholds, E., Schulte-Merker, S., Walderich, B. & Plasterk, R.H. Target-selected inactivation of the zebrafish rag1 gene. Science 297, 99–102 (2002).

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