Long-term imaging of living adult zebrafish.

The zebrafish has become a widely used animal model due, in large part, to its accessibility to and usefulness for high-resolution optical imaging. Although zebrafish research has historically focused mostly on early development, in recent years the fish has increasingly been used to study regeneration, cancer metastasis, behavior and other processes taking place in juvenile and adult animals. However, imaging of live adult zebrafish is extremely challenging, with survival of adult fish limited to a few tens of minutes using standard imaging methods developed for zebrafish embryos and larvae. Here, we describe a new method for imaging intubated adult zebrafish using a specially designed 3D printed chamber for long-term imaging of adult zebrafish on inverted microscope systems. We demonstrate the utility of this new system by nearly day-long observation of neutrophil recruitment to a wound area in living double-transgenic adult casper zebrafish with fluorescently labeled neutrophils and lymphatic vessels, as well as intubating and imaging the same fish repeatedly. We also show that Mexican cavefish can be intubated and imaged in the same way, demonstrating this method can be used for long-term imaging of adult animals from diverse aquatic species.

Anatomy and development of the pectoral fin vascular network in the zebrafish.

The pectoral fins of teleost fish are analogous structures to human forelimbs, and the developmental mechanisms directing their initial growth and patterning are conserved between fish and tetrapods. The forelimb vasculature is crucial for limb function, and it appears to play important roles during development by promoting development of other limb structures, but the steps leading to its formation are poorly understood. In this study, we use high-resolution imaging to document the stepwise assembly of the zebrafish pectoral fin vasculature. We show that fin vascular network formation is a stereotyped, choreographed process that begins with the growth of an initial vascular loop around the pectoral fin. This loop connects to the dorsal aorta to initiate pectoral vascular circulation. Pectoral fin vascular development continues with concurrent formation of three elaborate vascular plexuses, one in the distal fin that develops into the fin-ray vasculature and two near the base of the fin in association with the developing fin musculature. Our findings detail a complex, yet highly choreographed, series of steps involved in the development of a complete, functional, organ-specific vascular network.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition.

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

Common factors regulating patterning of the nervous and vascular systems.

The vascular and the nervous systems of vertebrates share many features with similar and often overlapping anatomy. The parallels between these two systems extend to the molecular level, where recent work has identified ever-increasing similarities between the molecular mechanisms employed in the specification, differentiation, and patterning of both systems. This review discusses some of the most recent literature on this subject, with particular emphasis on the roles that the Ephrin, Semaphorin, Netrin, and Slit signaling pathways play in vascular development.

Live Imaging of Intracranial Lymphatics in the Zebrafish.

RATIONALE: The recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species. OBJECTIVE: Examine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present. METHODS AND RESULTS: Using high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally, we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics. CONCLUSIONS: This discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development and opens up new avenues for probing meningeal lymphatic function in health and disease.

In vivo dissection of Rhoa function in vascular development using zebrafish.

The small monomeric GTPase RHOA acts as a master regulator of signal transduction cascades by activating effectors of cellular signaling, including the Rho-associated protein kinases ROCK1/2. Previous in vitro cell culture studies suggest that RHOA can regulate many critical aspects of vascular endothelial cell (EC) biology, including focal adhesion, stress fiber formation, and angiogenesis. However, the specific in vivo roles of RHOA during vascular development and homeostasis are still not well understood. In this study, we examine the in vivo functions of RHOA in regulating vascular development and integrity in zebrafish. We use zebrafish RHOA-ortholog (rhoaa) mutants, transgenic embryos expressing wild type, dominant negative, or constitutively active forms of rhoaa in ECs, pharmacological inhibitors of RHOA and ROCK1/2, and Rock1 and Rock2a/b dgRNP-injected zebrafish embryos to study the in vivo consequences of RHOA gain- and loss-of-function in the vascular endothelium. Our findings document roles for RHOA in vascular integrity, developmental angiogenesis, and vascular morphogenesis in vivo, showing that either too much or too little RHOA activity leads to vascular dysfunction.

To be or not to be: endothelial cell plasticity in development, repair, and disease.

Endothelial cells display an extraordinary plasticity both during development and throughout adult life. During early development, endothelial cells assume arterial, venous, or lymphatic identity, while selected endothelial cells undergo additional fate changes to become hematopoietic progenitor, cardiac valve, and other cell types. Adult endothelial cells are some of the longest-lived cells in the body and their participation as stable components of the vascular wall is critical for the proper function of both the circulatory and lymphatic systems, yet these cells also display a remarkable capacity to undergo changes in their differentiated identity during injury, disease, and even normal physiological changes in the vasculature. Here, we discuss how endothelial cells become specified during development as arterial, venous, or lymphatic endothelial cells or convert into hematopoietic stem and progenitor cells or cardiac valve cells. We compare findings from in vitro and in vivo studies with a focus on the zebrafish as a valuable model for exploring the signaling pathways and environmental cues that drive these transitions. We also discuss how endothelial plasticity can aid in revascularization and repair of tissue after damage- but may have detrimental consequences under disease conditions. By better understanding endothelial plasticity and the mechanisms underlying endothelial fate transitions, we can begin to explore new therapeutic avenues.

Development of the larval lymphatic system in zebrafish.

The lymphatic vascular system is a hierarchically organized complex network essential for tissue fluid homeostasis, immune trafficking and absorption of dietary fats in the human body. Despite its importance, the assembly of the lymphatic network is still not fully understood. The zebrafish is a powerful model organism that enables study of lymphatic vessel development using high-resolution imaging and sophisticated genetic and experimental manipulation. Although several studies have described early lymphatic development in the fish, lymphatic development at later stages has not been completely elucidated. In this study, we generated a new Tg(mrc1a:egfp)y251 transgenic zebrafish that uses a mannose receptor, C type 1 (mrc1a) promoter to drive strong EGFP expression in lymphatic vessels at all stages of development and in adult zebrafish. We used this line to describe the assembly of the major vessels of the trunk lymphatic vascular network, including the later-developing collateral cardinal, spinal, superficial lateral and superficial intersegmental lymphatics. Our results show that major trunk lymphatic vessels are conserved in the zebrafish, and provide a thorough and complete description of trunk lymphatic vessel assembly.

Interactions between mural cells and endothelial cells stabilize the developing zebrafish dorsal aorta.

Mural cells (vascular smooth muscle cells and pericytes) play an essential role in the development of the vasculature, promoting vascular quiescence and long-term vessel stabilization through their interactions with endothelial cells. However, the mechanistic details of how mural cells stabilize vessels are not fully understood. We have examined the emergence and functional role of mural cells investing the dorsal aorta during early development using the zebrafish. Consistent with previous literature, our data suggest that cells ensheathing the dorsal aorta emerge from a sub-population of cells in the adjacent sclerotome. Inhibition of mural cell recruitment to the dorsal aorta through disruption of pdgfr signaling leads to a reduced vascular basement membrane, which in turn results in enhanced dorsal aorta vessel elasticity and failure to restrict aortic diameter. Our results provide direct in vivo evidence for a functional role for mural cells in patterning and stabilization of the early vasculature through production and maintenance of the vascular basement membrane to prevent abnormal aortic expansion and elasticity.

Reck enables cerebrovascular development by promoting canonical Wnt signaling.

The cerebral vasculature provides the massive blood supply that the brain needs to grow and survive. By acquiring distinctive cellular and molecular characteristics it becomes the blood-brain barrier (BBB), a selectively permeable and protective interface between the brain and the peripheral circulation that maintains the extracellular milieu permissive for neuronal activity. Accordingly, there is great interest in uncovering the mechanisms that modulate the formation and differentiation of the brain vasculature. By performing a forward genetic screen in zebrafish we isolated no food for thought (nft (y72)), a recessive late-lethal mutant that lacks most of the intracerebral central arteries (CtAs), but not other brain blood vessels. We found that the cerebral vascularization deficit of nft (y72) mutants is caused by an inactivating lesion in reversion-inducing cysteine-rich protein with Kazal motifs [reck; also known as suppressor of tumorigenicity 15 protein (ST15)], which encodes a membrane-anchored tumor suppressor glycoprotein. Our findings highlight Reck as a novel and pivotal modulator of the canonical Wnt signaling pathway that acts in endothelial cells to enable intracerebral vascularization and proper expression of molecular markers associated with BBB formation. Additional studies with cultured endothelial cells suggest that, in other contexts, Reck impacts vascular biology via the vascular endothelial growth factor (VEGF) cascade. Together, our findings have broad implications for both vascular and cancer biology.

Growth Differentiation Factor 6 Promotes Vascular Stability by Restraining Vascular Endothelial Growth Factor Signaling.

OBJECTIVE: The assembly of a functional vascular system requires a coordinated and dynamic transition from activation to maturation. High vascular endothelial growth factor activity promotes activation, including junction destabilization and cell motility. Maturation involves junctional stabilization and formation of a functional endothelial barrier. The identity and mechanism of action of prostabilization signals are still mostly unknown. Bone morphogenetic protein receptors and their ligands have important functions during embryonic vessel assembly and maturation. Previous work has suggested a role for growth differentiation factor 6 (GDF6; bone morphogenetic protein 13) in vascular integrity although GDF6's mechanism of action was not clear. Therefore, we sought to further explore the requirement for GDF6 in vascular stabilization. APPROACH AND RESULTS: We investigated the role of GDF6 in promoting endothelial vascular integrity in vivo in zebrafish and in cultured human umbilical vein endothelial cells in vitro. We report that GDF6 promotes vascular integrity by counteracting vascular endothelial growth factor activity. GDF6-deficient endothelium has increased vascular endothelial growth factor signaling, increased vascular endothelial-cadherin Y658 phosphorylation, vascular endothelial-cadherin delocalization from cell-cell interfaces, and weakened endothelial cell adherence junctions that become prone to vascular leak. CONCLUSIONS: Our results suggest that GDF6 promotes vascular stabilization by restraining vascular endothelial growth factor signaling. Understanding how GDF6 affects vascular integrity may help to provide insights into hemorrhage and associated vascular pathologies in humans.

Single-cell analysis of endothelial morphogenesis in vivo.

Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo.

SNPfisher: tools for probing genetic variation in laboratory-reared zebrafish.

Single nucleotide polymorphisms (SNPs) are the benchmark molecular markers for modern genomics. Until recently, relatively few SNPs were known in the zebrafish genome. The use of next-generation sequencing for the positional cloning of zebrafish mutations has increased the number of known SNP positions dramatically. Still, the identified SNP variants remain under-utilized, owing to scant annotation of strain specificity and allele frequency. To address these limitations, we surveyed SNP variation in three common laboratory zebrafish strains using whole-genome sequencing. This survey identified an average of 5.04 million SNPs per strain compared with the Zv9 reference genome sequence. By comparing the three strains, 2.7 million variants were found to be strain specific, whereas the remaining variants were shared among all (2.3 million) or some of the strains. We also demonstrate the broad usefulness of our identified variants by validating most in independent populations of the same laboratory strains. We have made all of the identified SNPs accessible through 'SNPfisher', a searchable online database (snpfisher.nichd.nih.gov). The SNPfisher website includes the SNPfisher Variant Reporter tool, which provides the genomic position, alternate allele read frequency, strain specificity, restriction enzyme recognition site changes and flanking primers for all SNPs and Indels in a user-defined gene or region of the zebrafish genome. The SNPfisher site also contains links to display our SNP data in the UCSC genome browser. The SNPfisher tools will facilitate the use of SNP variation in zebrafish research as well as vertebrate genome evolution.

Temporal-specific roles of Rac1 during vascular development and retinal angiogenesis.

Angiogenesis, the formation of new blood vessels by remodeling and growth of pre-existing vessels, is a highly orchestrated process that requires a tight balance between pro-angiogenic and anti-angiogenic factors and the integration of their corresponding signaling networks. The family of Rho GTPases, including RhoA, Rac1, and Cdc42, play a central role in many cell biological processes that involve cytoskeletal changes and cell movement. Specifically for Rac1, we have shown that excision of Rac1 using a Tie2-Cre animal line results in embryonic lethality in midgestation (embryonic day (E) 9.5), with multiple vascular defects. However, Tie2-Cre can be also expressed during vasculogenesis, prior to angiogenesis, and is active in some hematopoietic precursors that can affect vessel formation. To circumvent these limitations, we have now conditionally deleted Rac1 in a temporally controlled and endothelial-restricted fashion using Cdh5(PAC)-iCreERT2 transgenic mice. In this highly controlled experimental in vivo system, we now show that Rac1 is required for embryonic vascular integrity and angiogenesis, and for the formation of superficial and deep vascular networks in the post-natal developing retina, the latter involving a novel specific function for Rac1 in vertical blood vessel sprouting. Aligned with these findings, we show that RAC1 is spatially involved in endothelial cell migration, invasion, and radial sprouting activities in 3D collagen matrix in vitro models. Hence, Rac1 and its downstream molecules may represent potential anti-angiogeneic therapeutic targets for the treatment of many human diseases that involve aberrant neovascularization and blood vessel overgrowth.

Aminoacyl-Transfer RNA Synthetase Deficiency Promotes Angiogenesis via the Unfolded Protein Response Pathway.

OBJECTIVE: Understanding the mechanisms regulating normal and pathological angiogenesis is of great scientific and clinical interest. In this report, we show that mutations in 2 different aminoacyl-transfer RNA synthetases, threonyl tRNA synthetase (tars(y58)) or isoleucyl tRNA synthetase (iars(y68)), lead to similar increased branching angiogenesis in developing zebrafish. APPROACH AND RESULTS: The unfolded protein response pathway is activated by aminoacyl-transfer RNA synthetase deficiencies, and we show that unfolded protein response genes atf4, atf6, and xbp1, as well as the key proangiogenic ligand vascular endothelial growth factor (vegfaa), are all upregulated in tars(y58) and iars(y68) mutants. Finally, we show that the protein kinase RNA-like endoplasmic reticulum kinase-activating transcription factor 4 arm of the unfolded protein response pathway is necessary for both the elevated vegfaa levels and increased angiogenesis observed in tars(y58) mutants. CONCLUSIONS: Our results suggest that endoplasmic reticulum stress acts as a proangiogenic signal via unfolded protein response pathway-dependent upregulation of vegfaa.

CBFß and RUNX1 are required at 2 different steps during the development of hematopoietic stem cells in zebrafish.

CBFß and RUNX1 form a DNA-binding heterodimer and are both required for hematopoietic stem cell (HSC) generation in mice. However, the exact role of CBFß in the production of HSCs remains unclear. Here, we generated and characterized 2 zebrafish cbfb null mutants. The cbfb(-/-) embryos underwent primitive hematopoiesis and developed transient erythromyeloid progenitors, but they lacked definitive hematopoiesis. Unlike runx1 mutants, in which HSCs are not formed, nascent, runx1(+)/c-myb(+) HSCs were formed in cbfb(-/-) embryos. However, the nascent HSCs were not released from the aorta-gonad-mesonephros (AGM) region, as evidenced by the accumulation of runx1(+) cells in the AGM that could not enter circulation. Moreover, wild-type embryos treated with an inhibitor of RUNX1-CBFß interaction, Ro5-3335, phenocopied the hematopoietic defects in cbfb(-/-) mutants, rather than those in runx1(-/-) mutants. Finally, we found that cbfb was downstream of the Notch pathway during HSC development. Our data suggest that runx1 and cbfb are required at 2 different steps during early HSC development. CBFß is not required for nascent HSC emergence but is required for the release of HSCs from AGM into circulation. Our results also indicate that RUNX1 can drive the emergence of nascent HSCs in the AGM without its heterodimeric partner CBFß.

Endothelial cilia are essential for developmental vascular integrity in zebrafish.

The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2, one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.

SoxF factors and Notch regulate nr2f2 gene expression during venous differentiation in zebrafish.

Initial embryonic determination of artery or vein identity is regulated by genetic factors that work in concert to specify the endothelial cell׳s (EC) fate, giving rise to two structurally unique components of the circulatory loop. The Shh/VEGF/Notch pathway is critical for arterial specification, while the orphan receptor nr2f2 (COUP-TFII) has been implicated in venous specification. Studies in mice have shown that nr2f2 is expressed in venous but not arterial ECs, and that it preferentially induces markers of venous cell fate. We have examined the role of nr2f2 during early arterial-venous development in the zebrafish trunk. We show that expression of a subset of markers of venous endothelial identity requires nr2f2, while the expression of nr2f2 itself requires sox7 and sox18 gene function. However, while sox7 and sox18 are expressed in both the cardinal vein and the dorsal aorta during early trunk development, nr2f2 is expressed only in the cardinal vein. We show that Notch signaling activity present in the dorsal aorta suppresses expression of nr2f2, restricting nr2f2-dependent promotion of venous differentiation to the cardinal vein.

The metalloproteinase inhibitor Reck is essential for zebrafish DRG development.

The neural crest is a migratory, multipotent cell lineage that contributes to myriad tissues, including sensory neurons and glia of the dorsal root ganglia (DRG). To identify genes affecting cell fate specification in neural crest, we performed a forward genetic screen for mutations causing DRG deficiencies in zebrafish. This screen yielded a mutant lacking all DRG, which we named sensory deprived (sdp). We identified a total of four alleles of sdp, all of which possess lesions in the gene coding for reversion-inducing cysteine-rich protein containing Kazal motifs (Reck). Reck is an inhibitor of metalloproteinases previously shown to regulate cell motility. We found reck function to be both necessary for DRG formation and sufficient to rescue the sdp phenotype. reck is expressed in neural crest cells and is required in a cell-autonomous fashion for appropriate sensory neuron formation. In the absence of reck function, sensory neuron precursors fail to migrate to the position of the DRG, suggesting that this molecule is crucial for proper migration and differentiation.