In vivo imaging of embryonic vascular development using transgenic zebrafish

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.     

3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.     

Platelet-Derived Growth Factor Receptor β Is Critical for Zebrafish Intersegmental Vessel Formation

Background

Platelet-derived growth factor receptor β (PDGFRβ) is a tyrosine kinase receptor known to affect vascular development. The zebrafish is an excellent model to study specific regulators of vascular development, yet the role of PDGF signaling has not been determined in early zebrafish embryos. Furthermore, vascular mural cells, in which PDGFRβ functions cell autonomously in other systems, have not been identified in zebrafish embryos younger than 72 hours post fertilization.

Methodology/Principal Findings

In order to investigate the role of PDGFRβ in zebrafish vascular development, we cloned the highly conserved zebrafish homolog of PDGFRβ. We found that pdgfrβ is expressed in the hypochord, a developmental structure that is immediately dorsal to the dorsal aorta and potentially regulates blood vessel development in the zebrafish. Using a PDGFR tyrosine kinase inhibitor, a morpholino oligonucleotide specific to PDGFRβ, and a dominant negative PDGFRβ transgenic line, we found that PDGFRβ is necessary for angiogenesis of the intersegmental vessels.

Significance/Conclusion

Our data provide the first evidence that PDGFRβ signaling is required for zebrafish angiogenesis. We propose a novel mechanism for zebrafish PDGFRβ signaling that regulates vascular angiogenesis in the absence of mural cells.     

Neurovascular development in the embryonic zebrafish hindbrain

The brain is made of billions of highly metabolically active neurons whose activities provide the seat for cognitive, affective, sensory and motor functions. The cerebral vasculature meets the brain's unusually high demand for oxygen and glucose by providing it with the largest blood supply of any organ. Accordingly, disorders of the cerebral vasculature, such as congenital vascular malformations, stroke and tumors, compromise neuronal function and survival and often have crippling or fatal consequences. Yet, the assembly of the cerebral vasculature is a process that remains poorly understood. Here we exploit the physical and optical accessibility of the zebrafish embryo to characterize cerebral vascular development within the embryonic hindbrain. We find that this process is primarily driven by endothelial cell migration and follows a two-step sequence. First, perineural vessels with stereotypical anatomies are formed along the ventro-lateral surface of the neuroectoderm. Second, angiogenic sprouts derived from a subset of perineural vessels migrate into the hindbrain to form the intraneural vasculature. We find that these angiogenic sprouts reproducibly penetrate into the hindbrain via the rhombomere centers, where differentiated neurons reside, and that specific rhombomeres are invariably vascularized first. While the anatomy of intraneural vessels is variable from animal to animal, some aspects of the connectivity of perineural and intraneural vessels occur reproducibly within particular hindbrain locales. Using a chemical inhibitor of VEGF signaling we determine stage-specific requirements for this pathway in the formation of the hindbrain vasculature. Finally, we show that a subset of hindbrain vessels is aligned and/or in very close proximity to stereotypical neuron clusters and axon tracts. Using endothelium-deficient cloche mutants we show that the endothelium is dispensable for the organization and maintenance of these stereotypical neuron clusters and axon tracts in the early hindbrain. However, the cerebellum's upper rhombic lip and the optic tectum are abnormal in clo. Overall, this study provides a detailed, multi-stage characterization of early zebrafish hindbrain neurovascular development with cellular resolution up to the third day of age. This work thus serves as a useful reference for the neurovascular characterization of mutants, morphants and drug-treated embryos.     

Arterial-venous network formation during brain vascularization involves hemodynamic regulation of chemokine signaling

During angiogenic sprouting, newly forming blood vessels need to connect to the existing vasculature in order to establish a functional circulatory loop. Previous studies have implicated genetic pathways, such as VEGF and Notch signaling, in controlling angiogenesis. We show here that both pathways similarly act during vascularization of the zebrafish central nervous system. In addition, we find that chemokine signaling specifically controls arterial-venous network formation in the brain. Zebrafish mutants for the chemokine receptor cxcr4a or its ligand cxcl12b establish a decreased number of arterial-venous connections, leading to the formation of an unperfused and interconnected blood vessel network. We further find that expression of cxcr4a in newly forming brain capillaries is negatively regulated by blood flow. Accordingly, unperfused vessels continue to express cxcr4a, whereas connection of these vessels to the arterial circulation leads to rapid downregulation of cxcr4a expression and loss of angiogenic characteristics in endothelial cells, such as filopodia formation. Together, our findings indicate that hemodynamics, in addition to genetic pathways, influence vascular morphogenesis by regulating the expression of a proangiogenic factor that is necessary for the correct pathfinding of sprouting brain capillaries.     

Plumbing the mysteries of vascular development using the zebrafish

The zebrafish has recently emerged as an advantageous model organism for studying how the stereotypic and evolutionarily conserved network of vertebrate blood vessels arises during development. The ability to screen for vascular-specific mutants and to image and experimentally manipulate blood vessels throughout living embryos has already yielded new insights into the anatomy of the early vasculature, the dynamics of growing blood vessels, the specification of early vascular progenitors, and arterial-venous differentiation of blood vessels.     

Notch signaling is required for arterial-venous differentiation during embryonic vascular development

Recent evidence indicates that acquisition of artery or vein identity during vascular development is governed, in part, by genetic mechanisms. The artery-specific expression of a number of Notch signaling genes in mouse and zebrafish suggests that this pathway may play a role in arterial-venous cell fate determination during vascular development. We show that loss of Notch signaling in zebrafish embryos leads to molecular defects in arterial-venous differentiation, including loss of artery-specific markers and ectopic expression of venous markers within the dorsal aorta. Conversely, we find that ectopic activation of Notch signaling leads to repression of venous cell fate. Finally, embryos lacking Notch function exhibit defects in blood vessel formation similar to those associated with improper arterial-venous specification. Our results suggest that Notch signaling is required for the proper development of arterial and venous blood vessels, and that a major role of Notch signaling in blood vessels is to repress venous differentiation within developing arteries. Movies available on-line     

Three-dimensional analysis of vascular development in the mouse embryo

Key vasculogenic (de-novo vessel forming) and angiogenic (vessel remodelling) events occur in the mouse embryo between embryonic days (E) 8.0 and 10.0 of gestation, during which time the vasculature develops from a simple circulatory loop into a complex, fine structured, three-dimensional organ. Interpretation of vascular phenotypes exhibited by signalling pathway mutants has historically been hindered by an inability to comprehensively image the normal sequence of events that shape the basic architecture of the early mouse vascular system. We have employed Optical Projection Tomography (OPT) using frequency distance relationship (FDR)-based deconvolution to image embryos immunostained with the endothelial specific marker PECAM-1 to create a high resolution, three-dimensional atlas of mouse vascular development between E8.0 and E10.0 (5 to 30 somites). Analysis of the atlas has provided significant new information regarding normal development of intersomitic vessels, the perineural vascular plexus, the cephalic plexus and vessels connecting the embryonic and extraembryonic circulation. We describe examples of vascular remodelling that provide new insight into the mechanisms of sprouting angiogenesis, vascular guidance cues and artery/vein identity that directly relate to phenotypes observed in mouse mutants affecting vascular development between E8.0 and E10.0. This atlas is freely available at http://www.mouseimaging.ca/research/mouse_atlas.html and will serve as a platform to provide insight into normal and abnormal vascular development.     

Microanatomy of adult zebrafish extraocular muscles

Binocular vision requires intricate control of eye movement to align overlapping visual fields for fusion in the visual cortex, and each eye is controlled by 6 extraocular muscles (EOMs). Disorders of EOMs are an important cause of symptomatic vision loss. Importantly, EOMs represent specialized skeletal muscles with distinct gene expression profile and susceptibility to neuromuscular disorders. We aim to investigate and describe the anatomy of adult zebrafish extraocular muscles (EOMs) to enable comparison with human EOM anatomy and facilitate the use of zebrafish as a model for EOM research. Using differential interference contrast (DIC), epifluorescence microscopy, and precise sectioning techniques, we evaluate the anatomy of zebrafish EOM origin, muscle course, and insertion on the eye. Immunofluorescence is used to identify components of tendons, basement membrane and neuromuscular junctions (NMJs), and to analyze myofiber characteristics. We find that adult zebrafish EOM insertions on the globe parallel the organization of human EOMs, including the close proximity of specific EOM insertions to one another. However, analysis of EOM origins reveals important differences between human and zebrafish, such as the common rostral origin of both oblique muscles and the caudal origin of the lateral rectus muscles. Thrombospondin 4 marks the EOM tendons in regions that are highly innervated, and laminin marks the basement membrane, enabling evaluation of myofiber size and distribution. The NMJs appear to include both en plaque and en grappe synapses, while NMJ density is much higher in EOMs than in somatic muscles. In conclusion, zebrafish and human EOM anatomy are generally homologous, supporting the use of zebrafish for studying EOM biology. However, anatomic differences exist, revealing divergent evolutionary pressures.     

Visualizing the cell-cycle progression of endothelial cells in zebrafish

The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.     

Semaphorin-plexin signaling guides patterning of the developing vasculature

Major vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. We show that proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and we identify mutations in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis.     

Identification of Inter-Organ Vascular Network: Vessels Bridging between Organs

Development and homeostasis of organs and whole body is critically dependent on the circulatory system. In particular, the circulatory system, the railways shuttling oxygen and nutrients among various organs, is indispensible for inter-organ humoral communication. Since the modern view of the anatomy and mechanics of the circulatory system was established in 17^th century, it has been assumed that humoral factors are carried to and from organs via vascular branches of the central arteries and veins running along the body axis. Over the past few decades, major advances have been made in understanding molecular and cellular mechanisms underlying the vascularization of organs. However, very little is known about how each organ is linked by vasculature (i.e., inter-organ vascular networks). In fact, the exact anatomy of inter-organ vascular networks has remained obscure. Herein, we report the identification of four distinct vessels, V1^LP, V2^LP, V3^LP and V4^LP, that bridge between two organs, liver and pancreas in developing zebrafish. We found that these inter-organ vessels can be classified into two types: direct and indirect types. The direct type vessels are those that bridge between two organs via single distinct vessel, to which V1^LP and V2^LP vessels belong. The indirect type bridges between two organs via separate branches that emanate from a stem vessel, and V3^LP and V4^LP vessels belong to this type. Our finding of V1^LP, V2^LP, V3^LP and V4^LP vessels provides the proof of the existence of inter-organ vascular networks. These and other yet-to-be-discovered inter-organ vascular networks may facilitate the direct exchange of humoral factors that are necessary for the coordinated growth, differentiation and homeostasis of the connected organs. It is also possible that the inter-organ vessels serve as tracks for their connected organs to follow during their growth to establish their relative positions and size differences.     

Mutant-specific gene expression profiling identifies SRY-related HMG box 11b (SOX11b) as a novel regulator of vascular development in zebrafish

Previous studies have identified two zebrafish mutants, cloche and groom of cloche, which lack the majority of the endothelial lineage at early developmental stages. However, at later stages, these avascular mutant embryos generate rudimentary vessels, indicating that they retain the ability to generate endothelial cells despite this initial lack of endothelial progenitors. To further investigate molecular mechanisms that allow the emergence of the endothelial lineage in these avascular mutant embryos, we analyzed the gene expression profile using microarray analysis on isolated endothelial cells. We find that the expression of the genes characteristic of the mesodermal lineages are substantially elevated in the kdrl ⁺ cells isolated from avascular mutant embryos. Subsequent validation and analyses of the microarray data identifies Sox11b, a zebrafish ortholog of SRY-related HMG box 11 (SOX11), which have not previously implicated in vascular development. We further define the function sox11b during vascular development, and find that Sox11b function is essential for developmental angiogenesis in zebrafish embryos, specifically regulating sprouting angiogenesis. Taken together, our analyses illustrate a complex regulation of endothelial specification and differentiation during vertebrate development.     

Universal GFP reporter for the study of vascular development

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.     

Ontogeny and nutritional control of adipogenesis in zebrafish (Danio rerio)

The global obesity epidemic demands an improved understanding of the developmental and environmental factors regulating fat storage. Adipocytes serve as major sites of fat storage and as regulators of energy balance and inflammation. The optical transparency of developing zebrafish provides new opportunities to investigate mechanisms governing adipocyte biology, however zebrafish adipocytes remain uncharacterized. We have developed methods for visualizing zebrafish adipocytes in vivo by labeling neutral lipid droplets with Nile Red. Our results establish that neutral lipid droplets first accumulate in visceral adipocytes during larval stages and increase in number and distribution as zebrafish grow. We show that the cellular anatomy of zebrafish adipocytes is similar to mammalian white adipocytes and identify peroxisome-proliferator activated receptor γ and fatty acid binding protein 11a as markers of the zebrafish adipocyte lineage. By monitoring adipocyte development prior to neutral lipid deposition, we find that the first visceral preadipocytes appear in association with the pancreas shortly after initiation of exogenous nutrition. Zebrafish reared in the absence of food fail to form visceral preadipocytes, indicating that exogenous nutrition is required for adipocyte development. These results reveal homologies between zebrafish and mammalian adipocytes and establish the zebrafish as a new model for adipocyte research.     

Fli+ etsrp+ hemato-vascular progenitor cells proliferate at the lateral plate mesoderm during vasculogenesis in zebrafish

Background

Vasculogenesis, the de novo formation of blood vessels from precursor cells is critical for a developing embryo. However, the signals and events that dictate the formation of primary axial vessels remain poorly understood.

Methodology/Principal Findings

In this study, we use ets-related protein-1 (etsrp), which is essential for vascular development, to analyze the early stages of vasculogenesis in zebrafish. We found etsrp ⁺ cells of the head, trunk and tail follow distinct developmental sequences. Using a combination of genetic, molecular and chemical approaches, we demonstrate that fli ⁺ etsrp ⁺ hemato-vascular progenitors (FEVPs) are proliferating at the lateral plate mesoderm (LPM). The Shh-VEGF-Notch-Hey2 signaling pathway controls the proliferation process, and experimental modulation of single components of this pathway alters etsrp ⁺ cell numbers at the LPM.

Conclusions/Significance

This study for the first time defines factors controlling proliferation, and cell numbers of pre-migratory FEVPs in zebrafish.     

Vascular anatomy of the lateral musculature of the flathead,Platycephalus bassensis (Teleostei: Perciformes)

Summary The vascular anatomy of the lateral musculature of the flatheadPlatycephalus bassensis, was studied by scanning electron microscopy of corrosion casts. Arteries and veins showed an alternating pattern in neighbouring vertebral segments. The red muscle was supplied by five major branches of the intermuscular artery, and the white muscle by infrequent branches of the intermuscular artery, dorsal segmental artery and ventral segmental artery. Venous drainage of the red and white muscles broadly mimicked the arterial supply. The functional unit of the trunk vasculature can be considered as an artery, a vein and connecting fine blood vessels. There appear to be 2 over-lapping types leading to alternating clockwise and counter-clockwise flows of blood. Small satellite vessels were observed running parallel to most of the larger blood vessels. No anatomical A-V shunt vessels, or series vascular connections between the red and white muscle, were observed. The irregular, alternating adult system is postulated to have developed from an earlier system showing strict bilateral symmetry and equal arterial and venous development in each vertebral segment.     

Evolutionary Conservation in Retinoid Signalling and Metabolism

In accord with the notion that retinoid signalling is of centralimportance in vertebrate evolution, a number of its componentsare evolutionarily conserved. Retinoid X nuclear receptors (RXRs),which interact directly with a number of signalling pathways,are highly conserved among mammals, Xenopus, and chick. We havestudied RXRs in zebrafish and find that they are also very wellconserved with respect to amino acid sequence and function,compared to mammalian RXRs. However, zebrafish has additionalsubtypes (RXR and RXR) which are altered in structure and function.New information which has come to light since these were firstdescribed suggests ways in which these unique subtypes couldfine-tune retinoid signalling in zebrafish. We have performedphylogenetic analysis with the zebrafish RXRs and RXRs fromother species to try to understand the evolutionary relationshipsamong them. In addition, we have found a retinoic acid (RA)-inducible,RA-metabolizing cytochrome P450 (P450RAI/CYP26) which is evolutionarilyconserved among vertebrates and has an important role in controllingretinoid signalling by regulating the level of biologicallyavailable RA.     

Cellular and molecular analyses of vascular tube and lumen formation in zebrafish

Tube and lumen formation are essential steps in forming a functional vasculature. Despite their significance, our understanding of these processes remains limited, especially at the cellular and molecular levels. In this study, we analyze mechanisms of angioblast coalescence in the zebrafish embryonic midline and subsequent vascular tube formation. To facilitate these studies, we generated a transgenic line where EGFP expression is controlled by the zebrafish flk1 promoter. We find that angioblasts migrate as individual cells to form a vascular cord at the midline. This transient structure is stabilized by endothelial cell-cell junctions, and subsequently undergoes lumen formation to form a fully patent vessel. Downregulating the VEGF signaling pathway, while affecting the number of angioblasts, does not appear to affect their migratory behavior. Our studies also indicate that the endoderm, a tissue previously implicated in vascular development, provides a substratum for endothelial cell migration and is involved in regulating the timing of this process, but that it is not essential for the direction of migration. In addition, the endothelial cells in endodermless embryos form properly lumenized vessels, contrary to what has been previously reported in Xenopus and avian embryos. These studies provide the tools and a cellular framework for the investigation of mutations affecting vasculogenesis in zebrafish.