Our vasculature plays diverse and critical roles in homeostasis and disease. In recent decades, the use of zebrafish has driven our understanding of vascular development into new areas, identifying ...new genes and mechanisms controlling vessel formation and allowing unprecedented observation of the cellular and molecular events that shape the developing vasculature. Here, we highlight key mechanisms controlling formation of the zebrafish vasculature and investigate how knowledge from this highly tractable model system has informed our understanding of vascular disease in humans.
Hogan and Schulte-Merker present a review discussing formation of the zebrafish vasculature. They highlight both the genes and mechanisms controlling vessel formation and connect this to the use of this model system for the study of vascular disease.
Interactions of transcription factors (TFs) with DNA regulatory sequences, known as enhancers, specify cell identity during animal development. Unlike TFs, the origin and evolution of enhancers has ...been difficult to trace. We drove zebrafish and mouse developmental transcription using enhancers from an evolutionarily distant marine sponge. Some of these sponge enhancers are located in highly conserved microsyntenic regions, including an
enhancer in the
-
region. We found that
enhancers in humans and mice share a suite of TF binding motifs with sponges, and that they drive gene expression patterns similar to those of sponge and endogenous
enhancers in zebrafish. Our results suggest the existence of an ancient and conserved, yet flexible, genomic regulatory syntax that has been repeatedly co-opted into cell type-specific gene regulatory networks across the animal kingdom.
The formation of the vertebrate vasculature involves the acquisition of endothelial cell identities, sprouting, migration, remodeling and maturation of functional vessel networks. To understand the ...cellular and molecular processes that drive vascular development, live-imaging of dynamic cellular events in the zebrafish embryo have proven highly informative. This review focusses on recent advances, new tools and new insights from imaging studies in vascular cell biology using zebrafish as a model system.
Lymphatic vessels arise chiefly from preexisting embryonic veins. Genetic regulators of lymphatic fate are known, but how dynamic cellular changes contribute during the acquisition of lymphatic ...identity is not understood. We report the visualization of zebrafish lymphatic precursor cell dynamics during fate restriction. In the cardinal vein, cellular commitment is linked with the division of bipotential Prox1-positive precursor cells, which occurs immediately prior to sprouting angiogenesis. Following precursor division, identities are established asymmetrically in daughter cells; one daughter cell becomes lymphatic and progressively upregulates Prox1, and the other downregulates Prox1 and remains in the vein. Vegfc drives cell division and Prox1 expression in lymphatic daughter cells, coupling signaling dynamics with daughter cell fate restriction and precursor division.
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•Prox1 expression initiates in the zebrafish vein before secondary angiogenesis•Prox1 is essential for lymphangiogenesis in zebrafish•Bipotential precursors divide to generate venous and lymphatic endothelial cells•Vegfc concomitantly drives bipotential precursor division and Prox1 expression
Using live imaging, Koltowska and Lagendijk et al. identify bipotential Prox1-expressing precursor cells that divide to generate daughter cells with distinct identities. Following Vegfc-driven cell division, a venous daughter cell remains in the vein and a lymphatic daughter cell leaves, a process by which one vascular lineage derives from another.
Meningitis caused by infectious pathogens is associated with vessel damage and infarct formation, however the physiological cause is often unknown. Cryptococcus neoformans is a human fungal pathogen ...and causative agent of cryptococcal meningitis, where vascular events are observed in up to 30% of patients, predominantly in severe infection. Therefore, we aimed to investigate how infection may lead to vessel damage and associated pathogen dissemination using a zebrafish model that permitted noninvasive in vivo imaging. We find that cryptococcal cells become trapped within the vasculature (dependent on their size) and proliferate there resulting in vasodilation. Localised cryptococcal growth, originating from a small number of cryptococcal cells in the vasculature was associated with sites of dissemination and simultaneously with loss of blood vessel integrity. Using a cell-cell junction tension reporter we identified dissemination from intact blood vessels and where vessel rupture occurred. Finally, we manipulated blood vessel tension via cell junctions and found increased tension resulted in increased dissemination. Our data suggest that global vascular vasodilation occurs following infection, resulting in increased vessel tension which subsequently increases dissemination events, representing a positive feedback loop. Thus, we identify a mechanism for blood vessel damage during cryptococcal infection that may represent a cause of vascular damage and cortical infarction during cryptococcal meningitis.
The lymphatic vascular system develops from the pre-existing blood vasculature of the vertebrate embryo. New insights into lymphatic vascular development have recently been achieved with the use of ...alternative model systems, new molecular tools, novel imaging technologies and growing interest in the role of lymphatic vessels in human disorders. The signals and cellular mechanisms that facilitate the emergence of lymphatic endothelial cells from veins, guide migration through the embryonic environment, mediate interactions with neighbouring tissues and control vessel maturation are beginning to emerge. Here, we review the most recent advances in lymphatic vascular development, with a major focus on mouse and zebrafish model systems.
The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in ...Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1) and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease, respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA-binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell-autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome.
Vascular endothelial growth factors (VEGFs) control angiogenesis and lymphangiogenesis during development and in pathological conditions. In the zebrafish trunk, Vegfa controls the formation of ...intersegmental arteries by primary angiogenesis and Vegfc is essential for secondary angiogenesis, giving rise to veins and lymphatics. Vegfd has been largely thought of as dispensable for vascular development in vertebrates. Here, we generated a zebrafish vegfd mutant by genome editing. vegfd mutants display significant defects in facial lymphangiogenesis independent of vegfc function. Strikingly, we find that vegfc and vegfd cooperatively control lymphangiogenesis throughout the embryo, including during the formation of the trunk lymphatic vasculature. Interestingly, we find that vegfd and vegfc also redundantly drive artery hyperbranching phenotypes observed upon depletion of Flt1 or Dll4. Epistasis and biochemical binding assays suggest that, during primary angiogenesis, Vegfd influences these phenotypes through Kdr (Vegfr2) rather than Flt4 (Vegfr3). These data demonstrate that, rather than being dispensable during development, Vegfd plays context-specific indispensable and also compensatory roles during both blood vessel angiogenesis and lymphangiogenesis.
The VEGFC/VEGFR3 signaling pathway is essential for lymphangiogenesis (the formation of lymphatic vessels from pre-existing vasculature) during embryonic development, tissue regeneration and tumor ...progression. The recently identified secreted protein CCBE1 is indispensible for lymphangiogenesis during development. The role of CCBE1 orthologs is highly conserved in zebrafish, mice and humans with mutations in CCBE1 causing generalized lymphatic dysplasia and lymphedema (Hennekam syndrome). To date, the mechanism by which CCBE1 acts remains unknown. Here, we find that ccbe1 genetically interacts with both vegfc and vegfr3 in zebrafish. In the embryo, phenotypes driven by increased Vegfc are suppressed in the absence of Ccbe1, and Vegfc-driven sprouting is enhanced by local Ccbe1 overexpression. Moreover, Vegfc- and Vegfr3-dependent Erk signaling is impaired in the absence of Ccbe1. Finally, CCBE1 is capable of upregulating the levels of fully processed, mature VEGFC in vitro and the overexpression of mature VEGFC rescues ccbe1 loss-of-function phenotypes in zebrafish. Taken together, these data identify Ccbe1 as a crucial component of the Vegfc/Vegfr3 pathway in the embryo.
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