**8. Zebrafish models of angiogenesis**

Zebrafish have recently gained much attention as an angiogenesis model system. Zebrafish embryos develop outside of the uterus, which greatly facilitates imaging during development. Furthermore, pigmentation can be inhibited chemically, or by using non-pigmented strains, such that the embryos maintain transparency throughout development. Many eggs are produced each breeding cycle, zebrafish are relatively easy and cheap to maintain, compared to rodents, and their embryonic development is much faster. Additional benefits of zebrafish-based models include passive uptake of chemicals added to the water, which eliminate the invasive administration procedures which are commonly needed in rodents and their small size which means that all tissues can be oxygenated by passive uptake from the water during development, thus allowing researchers to study phenotypes of embryos with severely disrupted vasculature, which are embryonically lethal and therefore difficult to study in mice (Weinstein et al, 1995).

Because of these unique characteristics of the zebrafish embryo, several genes with essential functions in the vasculature have been discovered using this system. One approach which has yielded the identification and characterization of novel genes important for developmental angiogenesis and vascular maturation is to perform unbiased screens of mutant embryos generated by random mutagenesis induced either by radiation, chemicals or insertion of small genetic fragments (Gaiano et al., 1996; Haffter et al., 1996; Knapik et al., 1996). Such screens have in the past yielded information on human congenital disorders of which the genetic background was previously unknown (Alders et al, 2009), and have – combined with the ability of the embryos to passively take up chemicals added to the water – been used to screen for, and identify novel drugs which efficiently correct the pathological vascular phenotypes (Peterson et al, 2004). Such an approach is called chemical genetics, due to the screening of novel chemicals for therapeutic effects in genetically modified zebrafish models, and is today being frequently used as a discovery tool in the pharmaceutical industry.

Recently researchers have further expanded the benefit of zebrafish-based model systems by generating many transgenic zebrafish strains which express fluorescent markers in particular cell types, organs or tissues, including endothelial cells of the vasculature (Jensen et al, 2009). By continuous observation of such transgenic embryos under the microscope, it is possible to follow the dynamics of growing vessels during zebrafish development in real time.

Such studies, which are difficult to perform in mice due to their *in utero* development, have yielded valuable insights into the process of vasculogenesis, which is the formation of the first embryonic vessels – the aorta, cardinal vein and thoracic duct – and on the origin of blood cells as well as the mechanism by which blood flow is initiated (Herbert et al, 2009; Lida et al, 2010; Yaniv et al, 2006).

Fig. 14. Blood vessels in the *Fli1:EGFP* transgenic zebrafish line. In the *fli1:EGFP* transgenic zebrafish, the promoter drives EGFP expression specifically in the vasculature. The top image show EGFP positive blood vessels (green) in a 27 hours old embryo, on the bottom left is shown blood vessels in the adult retina and on the bottom right in the adult tail fin.

The zebrafish genome has been fully sequenced and annotated, which makes it easy to interfere with genes involved in vascular development by injecting specially designed synthetic RNA-like molecules called morpholinos. These morpholinos are designed to have a complementary sequence to a particular target mRNA to which they will anneal and thus specifically target that transcript for destruction. The addition of morpholic acid groups on the nucleotide backbone of morpholinos makes them stable inside the cell and

particular cell types, organs or tissues, including endothelial cells of the vasculature (Jensen et al, 2009). By continuous observation of such transgenic embryos under the microscope, it is possible to follow the dynamics of growing vessels during zebrafish development in real

Such studies, which are difficult to perform in mice due to their *in utero* development, have yielded valuable insights into the process of vasculogenesis, which is the formation of the first embryonic vessels – the aorta, cardinal vein and thoracic duct – and on the origin of blood cells as well as the mechanism by which blood flow is initiated (Herbert et al, 2009;

Fig. 14. Blood vessels in the *Fli1:EGFP* transgenic zebrafish line. In the *fli1:EGFP* transgenic

The zebrafish genome has been fully sequenced and annotated, which makes it easy to interfere with genes involved in vascular development by injecting specially designed synthetic RNA-like molecules called morpholinos. These morpholinos are designed to have a complementary sequence to a particular target mRNA to which they will anneal and thus specifically target that transcript for destruction. The addition of morpholic acid groups on the nucleotide backbone of morpholinos makes them stable inside the cell and

zebrafish, the promoter drives EGFP expression specifically in the vasculature. The top image show EGFP positive blood vessels (green) in a 27 hours old embryo, on the bottom left is shown blood vessels in the adult retina and on the bottom right in the

time.

adult tail fin.

Lida et al, 2010; Yaniv et al, 2006).

they are therefore not degraded along with their mRNA target (Nasevicius & Ekker, 2000). Morpholinos, as well as mature mRNA - if over-expression rather than inhibition of a particular gene is being investigated - can be injected in the yolk of newly fertilized 1-cell stage embryos, and will then be present in all cells during development. As the cell number increases, the concentration of morpholino per cell will gradually decrease, and most morpholinos will therefore only be effective for up to 4 days after fertilization. This period is – due to the fast development of the zebrafish embryo – usually more than sufficient for angiogenesis studies, and thus does usually not pose a restriction on the study.

Morpholino-mediated disruption of a particular gene product is called knock-down, due to its transient nature compared to permanent knock-outs, and is today a widely used method to study the involvement of particular genes in angiogenesis and vascular functions during zebrafish development.

By using a zebrafish strain with enhanced green fluorescent protein expressed in endothelial cells under the *fli1* promoter (Lawson & Weinstein, 2002) (Fig. 14), knock-down of VEGF leads to a concentration-dependent inhibition of angiogenesis which can be quantified already 24 hours after fertilization (Nasevicius et al, 2000). In mice, even heterozygous deletion of VEGF leads to early embryonic lethality (Carmeliet et al, 1996) and the developmental consequence of subtle concentration differences in this critical angiogenic factor can therefore only be studied in zebrafish. Thus, zebrafish have been used recently to show that VEGF is only important for arterial growth, whereas growth of veins are practically unaffected by reduced VEGF levels.
