The Zebrafish as a Model Organism for Studying Vessel Formation

The zebrafish is a useful model for studies of development in general, but it offers particular advantages for studies of blood vessel development, including the ability to isolate vascular-specific mutants by forward genetics and use optical imaging methods to visualize blood vessels within the living animal with very high resolution. Recently the zebrafish has begun to yield novel insights into mechanisms of blood vessel formation in vivo.

Cardiovascular Mutants in the Zebrafish

Among the mutants uncovered in the Tübingen and Boston screens were a large number with circulatory system defects. Mutations that significantly impair cardiovascular development are readily identifiable in zebrafish because of the transparency and small size of the developing zebrafish embryo, which permits easy visualization of the heart and blood vessels and allows the animal to receive enough oxygen by passive diffusion to survive for 4 to 5 days in the absence of a functional circulation. The cardiovascular mutants from these and subsequent mutant screens included a number of mutants specifically defective in the formation of blood vessels. Table I provides a list of some of the vascular-specific mutants that have been published to date, while Figure 2 illustrates the vascular defects associated with a few of these mutants. Although vascular mutants represent a relatively small subset of the mutants reported thus far, several large-scale genetic screens specifically targeting blood vessel mutants have recently been undertaken [9; also our unpublished results], and these new screens are now yielding many new vascular-specific loci, providing an important resource for future studies of vascular development in the fish.

Table I

Mutant phenotype

Gene

Reference cloche Lack endothelial and circulating blood cells schwentine Loss of angioblasts (endothelial cell precursors) and failure to undergo angiogenesis gridlock Lack trunk and tail circulation due to improper assembly of the dorsal and lateral aortae violet beauregarde Massive enlargement of central cranial vessels and improper arterial-venous connections plcg(y10) Deficient in VEGF-mediated angiogenesis and arterial differentiation kurzschluss Branchial arteries fail to form properly and arterial-

venous shunts lead to loss of circulation in the trunk Out-of-bounds Display premature sprouting and mispatterned growth of the trunk intersegmental vessels unknown

Zebrafish ortholog offlk 1

Development (1997) 124, 3S1-3S9. Curr. Biol. (2002) 12(16), 1405-1412.

Vascular bHLH factor related Science (2000) 287, 1820-1824. to mammalian HRT2

Alk1 (acvrl1) Phospholipase C-g1 unknown PlexnD1

Development (2002) 129, 3009-3019.

Development (1996) 123, 293-302. Development (2002) 129, 973-9S2.

Figure 2 Vascular-specific mutants in the zebrafish. A number of different mutants have been isolated in zebrafish with defects in blood vessel formation. Cloche mutants lack virtually all blood endothelium (A). In situ hybridization of wild type (top) and mutant (bottom) embryos with vascular endothelial-specific probes such as Fli-1 reveals a lack of vascular staining except in a small patch in the posterior trunk (24hpf embryos, lateral views). Violet beauregarde mutants have defects in cranial vessel patterning (B). Mutants (bottom) display highly enlarged primary cranial vessels and reduced perfusion of secondary cranial vessels compared to wild type (top) embryos, as shown in these angiographic images (2.5 dpf embryos, dorsal views of head). Gridlock mutants lack caudal circulation due to a defect in proper formation of the dorsal aorta C). Mutants (right) have a normal pattern of cranial vessels but lack perfusion of more caudal vessels in comparison to wild type (left) animals (2.5 dpf embryos, lateral views of head and anterior trunk). (see color insert)

Figure 2 Vascular-specific mutants in the zebrafish. A number of different mutants have been isolated in zebrafish with defects in blood vessel formation. Cloche mutants lack virtually all blood endothelium (A). In situ hybridization of wild type (top) and mutant (bottom) embryos with vascular endothelial-specific probes such as Fli-1 reveals a lack of vascular staining except in a small patch in the posterior trunk (24hpf embryos, lateral views). Violet beauregarde mutants have defects in cranial vessel patterning (B). Mutants (bottom) display highly enlarged primary cranial vessels and reduced perfusion of secondary cranial vessels compared to wild type (top) embryos, as shown in these angiographic images (2.5 dpf embryos, dorsal views of head). Gridlock mutants lack caudal circulation due to a defect in proper formation of the dorsal aorta C). Mutants (right) have a normal pattern of cranial vessels but lack perfusion of more caudal vessels in comparison to wild type (left) animals (2.5 dpf embryos, lateral views of head and anterior trunk). (see color insert)

Transgenesis and Vascular Imaging in the Zebrafish

The application of transgenic technology to zebrafish cardiovascular research has provided a number of powerful tools for in vivo vascular imaging. Transgenic zebrafish lines expressing green fluorescent protein [GFP or enhanced (E)GFP] within vascular endothelial cells have been particularly useful for studying the formation of the vascu-lature in vivo. A germ-line transgenic expressing EGFP in the vasculature was generated using the zebrafish fli1 promoter (Figure 3; fli1 is a transcription factor expressed in the presumptive hemangioblast lineage, and later restricted to vascular endothelium, cranial neural crest derivatives, and a small subset of myeloid derivatives). This line expresses EGFP at high levels in vascular endothe-lium, permitting very high resolution long-term time-lapse analysis of developing blood vessels in vivo. Multiphoton confocal time lapse imaging of Fli-EGFP transgenic zebrafish has enabled detailed analysis of both normal vascular development and defective vessel formation due to genetic or experimental perturbations. Lawson and

Weinstein [10] showed in vivo that growing blood vessels are extremely active, extending and retracting filopodial processes up to tens of microns in all directions. In a separate study Isogai et al. [11] used time-lapse imaging of Fli-EGFP animals to study how the earliest network of angiogenic vessels (consisting of intersegmental and para-chordal vessels) assembles in the trunk. This study revealed a novel "two-step" mechanism for vascular network formation, with a primary vascular network forming first from artery-derived sprouts followed by emergence of a set of vein-derived secondary sprouts that interact dynamically with the primary network to generate the final functional "wiring" of the trunk network. The results of this study showed that blood flow does not play an important role in the initial assembly and gross anatomical patterning of this network, but is likely a critical determinant of the interconnections between primary and secondary vessels and their eventual arterial-venous identity. The Fli-EGFP transgenic line also permits imaging of blood vessels in adult zebrafish, as revealed in another recent study, of vascular network reassembly in the regenerating zebrafish caudal fin [12].

Angiogenesis Isoquinolines Zebrafish

Figure 3 Blood vessels imaged in Fli-EGFP transgenic zebrafish. Vascular endothelial cells and their angioblast precursors are brightly green fluorescent in Fli-EGFP transgenic animals (A). Image shown is a confocal microangiogram of a 7 day-old transgenic zebrafish. Fine cellular details can be discerned, including thin filopodial processes extending from growing intersegmental vessels in the trunk (B) and central arteries assembling in the zebrafish hindbrain (C). Images shown are confocal microangiograms from approximately 1.5 day-old transgenic zebrafish. (see color insert)

Figure 3 Blood vessels imaged in Fli-EGFP transgenic zebrafish. Vascular endothelial cells and their angioblast precursors are brightly green fluorescent in Fli-EGFP transgenic animals (A). Image shown is a confocal microangiogram of a 7 day-old transgenic zebrafish. Fine cellular details can be discerned, including thin filopodial processes extending from growing intersegmental vessels in the trunk (B) and central arteries assembling in the zebrafish hindbrain (C). Images shown are confocal microangiograms from approximately 1.5 day-old transgenic zebrafish. (see color insert)

Studying Arterial-Venous Fate Determination Using the Zebrafish

One of the recent important contributions to the study of microvasculature using the zebrafish has been the identification of molecular pathways responsible for arterial-venous (A-V) differentiation of endothelial cells. In the past, the arterial-venous fate of endothelial cells was believed to follow from physiological parameters such as differences in blood flow and pressure, but recent work has shown that early endothelial arterial-venous differentiation is in fact genetically programmed. Initial evidence that arterial and venous endothelial cells possess distinct molecular identities came from work with ephrin and Eph genes in mice. Wang et al. [13] described the expression of ephrin B2 (Efnb2), a member of the ephrin family of membrane ligands. Prior to the onset of flow, Efnb2 is expressed specifically in arterial endothelial cells and is absent in venous endothelial cells, whereas the ephrin B2 receptor, Ephb4, is preferentially expressed in veins. Targeted gene deletion of each member of this ligand-receptor pair resulted in similar cardiovascular abnormalities, demonstrating their necessity, and likely direct interaction, for normal vascular development. Although these previous studies demonstrated the existence of functionally important molecular differences between arterial venous endothelial cells, they did not reveal how this fate choice was initially made. Zebrafish studies have been critical in uncovering and dissecting the functional roles of the upstream factors specifying arterial and venous endothe-lial cell fates, identifying a signaling cascade consisting of sequential hedgehog, vascular endothelial growth factor, and notch signaling. The discussion that follows reviews these findings.

A variety of studies in mammals and other vertebrates had revealed the specific expression of Notch signaling genes (Notch, Delta, Jagged, and so or) in arterial but not in venous endothelial cells. Murine knockout studies showed that these molecules play an important functional role in the vasculature, and their arterial-specific expression suggested they might be playing a specific role in artery formation. A number of recent studies in the zebrafish [14-16] have demonstrated that Notch signaling promotes arterial differentiation at the expense of venous differentiation during vascular development. Notch signaling was repressed in zebrafish embryos either genetically, using the neurogenic mindbomb (mib) mutant, or experimentally, by injecting mRNA encoding a dominant-negative DNA binding mutant of Xenopus suppressor of hairless protein. In either case, repression of Notch signaling resulted in loss of ephrinB2a expression from arteries and ectopic expansion of normally venous-restricted markers such as ephb4 and flt-4 into the arterial domain. Conversely, activation of Notch signaling [either by heat-shock promoter-driven ubiquitous expression of the Notch1a intracellular domain (Notch1a-ICD) or by Fli1-promoter driven vascular-specific expression of Notch5-ICD] suppressed expression of vein-restricted markers and promoted ectopic expression of ephrinB2a and other arterial markers in venous vessels. The vascular specific expression of Notch demonstrated the vascular endothelial cell-autonomy of Notch-ICD effects, confirming that Notch is in fact acting at the level of the vascular endothelial cell itself and not via indirect signals from some other, adjacent Notch-responsive cells or tissues.

Lawson and colleagues further dissected the A-V differentiation signaling hierarchy by demonstrating that sonic hedgehog (shh) and vascular endothelial growth factor (vegf) act upstream of Notch [15]. As in embryos lacking Notch signaling, embryos lacking shh or vegf fail to express ephrin-B2a within their blood vessels. Overexpression of shh promotes ectopic arterial vessel formation in the trunk, whereas overexpression of vegf via injection of vegf mRNA suppresses expression of vein-restricted markers and results in expression of ephrinB2a and other arterial markers in venous vessels. By combining activation or inhibition of each of the different signaling pathways in a series of "molecular epistasis" experiments, it was found that shh activity induces expression of vegf in the somites, which then activates notch signaling in the adjacent endothelial cells of the developing dorsal aorta, promoting arterial differentiation. Genetic screening methods were also used to identify genes functioning downstream from vegf in the zebrafish [17]. A zebrafish mutant was uncovered that was deficient in both angiogenesis and arterial differentiation as a result of a defect in phospholipase C gamma-1 (plcg1). Phospholipase C genes are known effectors of signaling via receptor tyrosine kinases such as the vegf receptor Flk1, and the vascular expression of plcg1 and vascular-specific phe-notype of the mutant in this gene suggested that it might be functioning downstream of vegf signaling. Indeed, further experiments showed that plcg1 mutants were insensitive to both angiogenic and arterial differentiation responses to vegf overexpression. In support of the zebrafish findings regarding roles for hedgehog and vegf signaling in the vasculature, recent studies in mice have also implicated shh and vegf signaling in regulating blood vessel growth and arterial differentiation [18-21], demonstrating the conservation of genetic programs between vertebrate species.

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