Accelerated gene and drug discovery – strategies for the future
The mouse genome contains between 25,000 to 35,000 genes, several of which are modified transcriptionally or posttranslationally. Thus, more than 100,000 gene products are thought to be relevant for the various biological processes in normal health and, when malfunctioning, may contribute to the pathogenesis of various disorders. Identifying such “disease candidate genes” and understanding their functional role in particular disease processes via “functional genomics” offers opportunities for the development of novel therapeutic strategies and drug candidates. Even though the genome of the mouse can be precisely manipulated via sophisticated techniques, and relevant disease mouse models can be generated that closely mimick human disease, and the mouse offers the advantage of studying the physiological implications of these genetic manipulation, such studies are time-consuming, expensive and labor-intensive. It has been speculated that genome-wide characterization of the function of all these genes would require a world-wide conserted effort for several decades, perhaps even more than a century. Alternative, more rapid, less expensive, and higher-throughput strategies are thus required.
Similar strategies are needed to accelerate drug discovery. Succesful therapy of complex human disorders, such as cancer and neurodegeneration, will require, in most cases, combination treatment with various drugs in order to increase the efficacy and minimize acquired drug resistance. This mandates the development of novel drugs with complementary mechanisms. Because combination therapy may increase toxicity, such drugs should have an acceptable safety profile. As explained below, small aquatic models offer novel opportunities to accelerate drug discovery as well.
Small vertebrate organisms have emerged as key players in the post-genomic era for the accelerated functional characterization of novel genes (“functional genomics”). Zebrafish embryos and Xenopus tadpoles in particular represent attractive and valuable models to rapidly identify and characterize novel genes and drug candidates involved in angiogenesis and lymphangiogenesis.

Development
Both animals can produce thousands of eggs on a weekly basis, which can be easily manipulated and used to study embryonic development. The larvae develop outside of the mother’s body, are transparent during the first phase of growth, and develop extremely fast –the heart starts beating within 24h in a zebrafish embryo and within 2 days in a tadpole, and, already within four days, most blood vessels, lymph vessels and nerves are fully developed.

Rapid and easy genetic and pharmacological manipulation
These animal models also offer attractive opportunities for easy and rapid genetic manipulation. Historically, they were used in forward genetic screens whereby the fish were exposed to mutagenic substances to induce point mutations in genes, which were subsequently identified via time-consuming and labour-intensive cloning efforts. More recently, the morpholino technology made it possible to knockdown protein expression by injecting modified antisense “morpholino” oligonucleotides in the 1-4 cell stage embryo. This reverse genetics approach allows one to rapidly determine the function of a novel gene within days (not months or years, as in the mouse), and to study gene interactions (via knockdown of two or more genes) or gene-dosage dependent effects (via graded knockdown) which would require months / years of intercrossing in mice. Overexpression of a gene can also be readily performed, either by injecting in vitro transcribed RNA or by generating transgenic animals expression the gene of interest under a (tissue-specific) promoter. During the last year, transgenesis has become more efficient by using the Tol2-transposon mediated gene transfer in zebrafish, and by using the SceI-meganuclease technique in tadpoles. Another significant advantage of these aquatic animal models is that they can be readily exposed to a desired concentration of chemical compounds (pharmacological inhibitors or activators alike) at defined stages of embryonic development, thereby allowing easy, rapid and inexpensive inducible gene-dosage dependent inhibition (or alteration) of gene function and providing a competetive alternative option to the expensive, time-consuming and labour-intensive conditional gene-(in)activation in mice. Overall, the reduced costs, accelerated time frame, and technical accessibility associated with using these genetic aquatic animal models allows one to study >10-fold more gene candidates than in the mouse.

Drug discovery and conditional genetics via chemico-genetics
Recently, small animal models have also been used to identify novel chemical compounds with a strong therapeutic potential. Indeed, numerous pharmaceutical companies are currently devoting multimillion dollar budgets to developing other — in many cases orally deliverable — chemical anti- angiogenic drugs. There is thus a growing need to find reliable, cheap and fast assays to screen large numbers of chemical compounds in live animal models. Due to their unprecedented and widely applicable features as animal models, zebrafish and tadpole embryos have become invaluable tools for medium-throughput drug screens in pharmaceutical companies. The VRC is developing new transgenic lines of small aquatic animal models for such screening purposes.

Simple but sophisticated phenotypic analysis in the embryo
Characterization of the phenotypes of the genetically manipulated embryos can be performed by rapid, simple techniques, involving, for instance, live analysis and whole mount embryo stainings by in situ hybridization or immunohistochemistry using conventional microscopy. However, the recent availability of transgenic reporter lines, expressing fluorescent markers in specific vascular or neuronal cell types, in combination with the transparency of the embryos, and improved imaging techniques of offer unparalleled opportunities for high-resolution dynamic three-dimensional video imaging, using state-of-the-art multi-photon confocal microscopy. For instance, blood vessel formation can be dynamically analyzed for several days at a resolution, allowing the visualization of single endothelial cell filopodia. In addition, colored or fluorescent dyes can also be microinjected in these frog and fish larvae to study the vascular system by angiography (a dye is injected in the heart of a living embryo) or lymphangiography (injection of dye in the tail), or charaterize axonal projections in the nervous sytem. In fact, these small aquatic animals are the only models thus far available to study live outgrowth and formation of the vascular network and offers great advantages over the static analysis of vessel development in the mouse. Apart from these sophisticated physiological assays, analysis of the molecular pathways can be readily dissected by classical molecular and biochemical techniques. Our recent publications indicate that these aquatic animal models have great predictive value for gene and drug discovery in mice, i.e. genes identified and drug lead compounds identified in fish or frogs had similar biological or therapeutic importance in mice. 

Disease models in the adult
Almost 20 years ago, the mouse was the preferred genetic animal model – interest in this model, however, exponentially increased when disease models in adult mice were developed. Aquatic animal models face a similar challenge and promise. Tumor models have already been documented in zebrafish. We are therefore setting up novel assays and disease models, including models of neurodegeneration, cancer and tissue regeneration – for instance, after amputation of the tail fin, angiogenesis can be studied in the regenerating tail fin (an assay which we routinely use to test the anti-angiogenic activity of new chemical compounds).

video: How Intersomitic Vessels develop in zebrafish
video: Lymphatic & Blood vessel flow
video: Beating Lymphatic Heart

Aquatic facility
The KULeuven aquatic facilities has been recently built close to our department and consist of two units, the first is the aquatic facility itself where two rooms are dedicated to the maintenance and reproduction of zebrafish, one room to Xenopus laevis and one to Xenopus tropicalis. The total capacity of the faclility is

The second unit consist of one microinjection lab for fish and one similar lab for Xenopus. Each lab is equiped with several microinjection stations and fluorescent microscopes for live imaging. The unit comprises in addition two small quarantine rooms* for sanitary transit of new animals.

The facility is run by KU-Leuven personnel consisting of one supervisor and two animal care takers with occasional veterinarian advice from the University of Gent (contact person: mailto: This e-mail address is being protected from spam bots, you need JavaScript enabled to view it , Proefdierencentrum KU-Leuven).

video: Virtual Tour in the zebrafish facility

video: Virtual Tour in the xenopus facility

 References

Ny A, Autiero M, Carmeliet P. Zebrafish and Xenopus tadpoles: small animal models to study angiogenesis and lymphangiogenesis. Exp Cell Res. 2006 Mar 10;312(5):684-93. Epub 2005 Nov 23. Review.

Ny A, Koch M, Schneider M, Neven E, Tong RT, Maity S, Fischer C, Plaisance S, Lambrechts D, Heligon C, Terclavers S, Ciesiolka M, Kalin R, Man WY, Senn I, Wyns S, Lupu F, Brandli A, Vleminckx K, Collen D, Dewerchin M, Conway EM, Moons L, Jain RK, Carmeliet P. A genetic Xenopus laevis tadpole model to study lymphangiogenesis. Nat Med. 2005 Sep;11(9):998-1004. Epub 2005 Aug 14.

Le Bras B, Barallobre MJ, Homman-Ludiye J, Ny A, Wyns S, Tammela T, Haiko P, Karkkainen MJ, Yuan L, Muriel MP, Chatzopoulou E, Breant C, Zalc B, Carmeliet P, Alitalo K, Eichmann A, Thomas JL.VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain. Nat Neurosci. 2006 Mar;9(3):340-8. Epub 2006 Feb 5.

De Smet F, Carmeliet P, Autiero M. Fishing and frogging for anti-angiogenic drugs. Nat Chem Biol. 2006 May;2(5):228-9. No abstract available.

Aerts S, Lambrechts D, Maity S, Van Loo P, Coessens B, De Smet F, Tranchevent LC, De Moor B, Marynen P, Hassan B, Carmeliet P, Moreau Y. Gene prioritization through genomic data fusion. Nat Biotechnol. 2006 May;24(5):537-44. Erratum in: Nat Biotechnol. 2006 Jun;24(6):719.

Autiero M, De Smet F, Claes F, Carmeliet P. Role of neural guidance signals in blood vessel navigation. Cardiovasc Res. 2005 Feb 15;65(3):629-38.

Lu X, Le Noble F, Yuan L, Jiang Q, De Lafarge B, Sugiyama D, Breant C, Claes F, De Smet F, Thomas JL, Autiero M, Carmeliet P, Tessier-Lavigne M, Eichmann A. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature. 2004 Nov 11;432(7014):179-86.

Chittenden TW, Claes F, Lanahan AA, Autiero M, Palac RT, Tkachenko EV, Elfenbein A, Ruiz de Almodovar C, Dedkov E, Tomanek R, Li W, Westmore M, Singh JP, Horowitz A, Mulligan-Kehoe MJ, Moodie KL, Zhuang ZW, Carmeliet P, Simons M. Selective regulation of arterial branching morphogenesis by synectin. Dev Cell. 2006 Jun;10(6):783-95.