Nascent vascular networks adapt to the increasing metabolic demands of growing tissues by expanding via angiogenesis. As vascular networks expand, blood vessels remodel, progressively refining vascular connectivity to generate a more haemodynamically efficient network. This is driven by interplay between endothelial cell (EC) signalling and blood flow. While much is known about angiogenesis, less is understood of how blood flow remodels vessels. Here we employ the zebrafish sub-intestinal venous plexus (SIVP) to characterise blood flow-dependent remodelling. Using live imaging to track ECs we show that blood flow controls SIVP remodelling by coordinating collective EC migration within the developing plexus. Blood flow opposes continuous ventral EC migration within the SIVP and is required for angiogenic sprout regression to support plexus growth. Sprout regression occurs by coordinated migration of ECs from non-perfused leading sprouts, which migrate against blood flow and incorporate into the sub-intestinal vein. Sprout regression remains compatible with low blood flow but requires vegfr3/flt4 function under these conditions. Collectively, these studies reveal how blood flow sculpts a developing vascular plexus by coordinating EC migration and balancing vascular remodelling via vegfr3/flt4.

Angiogenesis is a multi-factorial physiological process deregulated in human diseases characterised by excessive or insufficient blood vessel formation. Emerging evidence highlights a novel role for microRNAs as regulators of angiogenesis. Previous studies addressing the effect of miR-133a expression in endothelial cells during blood vessel formation have reported conflicting results. Here, we have assessed the specific effect of mature miR-133a strands in angiogenesis and the expression of endothelial angiogenic genes. Transfection of miR-133a-3p or -5p mimics in primary human endothelial cells significantly inhibited proliferation, migration, and tubular morphogenesis of transfected cells. Screening of gene arrays related to angiogenic processes, and further validation by TaqMan qPCR, revealed that aberrant expression of miR-133a-3p led to a decrease in the expression of genes encoding pro-angiogenic molecules, whilst increasing those with anti-angiogenic functions. Ingenuity Pathway Analysis of a collection of genes differentially expressed in cells harbouring miR-133a-3p, predicted decreased cellular functions related to vasculature branching and cell cycle progression, underlining the inhibitory role of miR-133a-3p in angiogenic cellular processes. Our results suggest that controlled delivery of miR-133a-3p mimics, or antagomirs in diseased endothelial cells, might open new therapeutic interventions to treat patients suffering from cardiovascular pathologies that occur with excessive or insufficient angiogenesis.

The mature vertebrate heart develops from a simple linear cardiac tube during early development through a series of highly asymmetric morphogenetic processes including cardiac looping and chamber ballooning. While the directionality of heart morphogenesis is partly controlled by embryonic laterality signals, previous studies have suggested that these extrinsic laterality cues interact with tissue-intrinsic signals in the heart to ensure robust asymmetric cardiac morphogenesis. Using live in vivo imaging of zebrafish embryos we describe a left-sided, chamber-specific expansion of the extracellular matrix (ECM) between the myocardium and endocardium at early stages of heart morphogenesis. We use Tomo-seq, a spatial transcriptomic approach, to identify transient and regionalised expression of hyaluronan and proteoglycan link protein 1a ( hapln1a), encoding an ECM cross-linking protein, in the heart tube prior to cardiac looping overlapping with regionalised ECM expansion. Loss- and gain-of-function experiments demonstrate that regionalised Hapln1a promotes heart morphogenesis through regional modulation of ECM thickness in the heart tube. Finally, we show that while induction of asymmetric hapln1a expression is independent of embryonic left-right asymmetry, these laterality cues are required to orient the hapln1a -expressing cells asymmetrically along the left-right axis of the heart tube. Together, we propose a model whereby laterality cues position hapln1a expression on the left of the heart tube, and this asymmetric Hapln1a deposition drives ECM asymmetry and subsequently promotes robust asymmetric cardiac morphogenesis.

Zebrafish allow unrivalled in vivo imaging of vascular development due to their optical translucency and the availability of transgenic lines which fluorescently label cells and tissues of interest. Advances in light sheet fluorescence microscopy allow longer and faster imaging of live embryos at higher resolutions than previously possible, which facilitates study of dynamic cellular and molecular mechanisms underlying vessel formation and function. Here we describe a workflow using lightsheet microscopy to quantify endothelial cell (EC) migration dynamics during vascular development. Tracking movement of EC nuclei and analyzing the properties of EC migration trajectories permit detailed studies of angiogenesis and vascular remodeling in different contexts.

Antimicrobial/anticancer peptides (AMPs/ACPs) have shown promising results as new therapeutic agents in cancer thearpy. Among them, the designed amphiphilic α-helical peptide G(IIKK)3I-NH2 (G3) displayed great affinity and specificity in targeting cancer cells. Here, we report new insights on how G3 penetrates cancer cells. G3 showed high specificity to HCT-116 colon cancer cells compared to the HDFs (human neonatal primary dermal fibroblasts) control. With high concentrations of peptide, a clear cancer cell membrane disruption was observed through SEM. Gene knockdown of the endocytic pathways demonstrated that an energy-dependent endocytic pathway is required for the uptake of the peptide. In addition, G3 can protect and selectively deliver siRNAs into cancer cells and successfully modulated their gene expression. Gene delivery was also tested in 3D cancer spheroids and showed deep penetration delivery into the cancer spheroids. Finally, the in vivo toxicity of G3 was evaluated on zebrafish embryos, showing an increasing toxicity effect with concentration. However, the toxicity of the peptide was attenuated when complexed with siRNA. In addition, negligible toxicity was observed at the concentration range for efficient gene delivery. The current results demonstrate that G3 is promising as an excellent agent for cancer therapy.

Several studies have shown a clear association between periodontal disease and increased risk of cardiovascular disease. Porphyromonas gingivalis (Pg), a key oral pathogen, and its cell surface‐expressed gingipains, induce oedema in a zebrafish larvae infection model although the mechanism of these vascular effects is unknown. Here, we aimed to determine whether Pg‐induced vascular damage is mediated by gingipains. In vitro, human endothelial cells from different vascular beds were invaded by wild‐type (W83) but not gingipain‐deficient (ΔK/R‐ab) Pg. W83 infection resulted in increased endothelial permeability as well as decreased cell surface abundance of endothelial adhesion molecules PECAM‐1 and VE‐cadherin compared to infection with ΔK/R‐ab. In agreement, when transgenic zebrafish larvae expressing fluorescently labelled PECAM‐1 or VE‐cadherin were systemically infected with W83 or ΔK/R‐ab, a significant reduction in adhesion molecule fluorescence was observed specifically in endothelium proximal to W83 bacteria through a gingipain‐dependent mechanism. Furthermore, this was associated with increased vascular permeability in vivo when assessed by dextran leakage microangiography. These data are the first to show that Pg directly mediates vascular damage in vivo by degrading PECAM‐1 and VE‐cadherin. Our data provide a molecular mechanism by which Pg might contribute to cardiovascular disease.

Neurovascular coupling (through which local cerebral blood flow changes in response to neural activation are mediated) is impaired in many diseases including diabetes. Current preclinical rodent models of neurovascular coupling rely on invasive surgery and instrumentation, but transgenic zebrafish coupled with advances in imaging techniques allow non-invasive quantification of cerebrovascular anatomy, neural activation, and cerebral vessel haemodynamics. We therefore established a novel non-invasive, non-anaesthetised zebrafish larval model of neurovascular coupling, in which visual stimulus evokes neuronal activation in the optic tectum that is associated with a specific increase in red blood cell speed in tectal blood vessels. We applied this model to the examination of the effect of glucose exposure on cerebrovascular patterning and neurovascular coupling. We found that chronic exposure of zebrafish to glucose impaired tectal blood vessel patterning and neurovascular coupling. The nitric oxide donor sodium nitroprusside rescued all these adverse effects of glucose exposure on cerebrovascular patterning and function. Our results establish the first non-mammalian model of neurovascular coupling, offering the potential to perform more rapid genetic modifications and high-throughput screening than is currently possible using rodents. Furthermore, using this zebrafish model, we reveal a potential strategy to ameliorate the effects of hyperglycemia on cerebrovascular function.

Cilia are essential for morphogenesis and maintenance of many tissues. Loss-of-function of cilia in early Zebrafish development causes a range of vascular defects, including cerebral hemorrhage and reduced arterial vascular mural cell coverage. In contrast, loss of endothelial cilia in mice has little effect on vascular development. We therefore used a conditional rescue approach to induce endothelial cilia ablation after early embryonic development and examined the effect on vascular development and mural cell development in postembryonic, juvenile, and adult Zebrafish.ift54(elipsa)-mutant Zebrafish are unable to form cilia. We rescued cilia formation and ameliorated the phenotype of ift54 mutants using a novel Tg(ubi:loxP-ift54-loxP-myr-mcherry,myl7:EGFP)sh488 transgene expressing wild-type ift54 flanked by recombinase sites, then used a Tg(kdrl:cre)s898 transgene to induce endothelial-specific inactivation of ift54 at postembryonic ages. Fish without endothelial ift54 function could survive to adulthood and exhibited no vascular defects. Endothelial inactivation of ift54 did not affect development of tagln-positive vascular mural cells around either the aorta or the caudal fin vessels, or formation of vessels after tail fin resection in adult animals.Endothelial cilia are not essential for development and remodeling of the vasculature in juvenile and adult Zebrafish when inactivated after embryogenesis.

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2 -dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.

Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development.

Introduction ATF2 also known as cyclic AMP response element binding protein 2 (CREB2) is a member of the leucine zipper (bZIP) transcription factor family that binds to specific DNA sequences and regulates the transcriptional activation of target genes. ATF2 regulation and functions have been studied in a number of developmental and pathological conditions. Studies have shown that activation of ATF2 by VEGF mediates angiogenic processes such as endothelial cell migration and tubular morphogenesis but the molecular role of ATF2 in these processes is largely unknown. To shed some light on this matter we are identifying ATF2-target genes induced by stimulation of endothelial cells with pro-angiogenic stimuli. Methods HUVEC were stimulated with various pro-angiogenic stimuli at different times, and the activation (phosphorylation) status of ATF2 was determined by Western Blot. ATF2 functionality in endothelial cells was suppressed by infecting HUVEC with an adenovirus encoding a phosphorylation-mutant, dominant-negative version of ATF2 (Ad-ATF2AA) where phosphorylation residues Thr69 and Thr71 have been mutated to Ala. HUVEC infection with an adenovirus encoding GFP was used as a control. PCR-based screening of specific gene arrays was used to identify the effect of ATF2 loss-of-function in the transcriptional expression of genes related to the NOTCH signalling pathway. The screening results were validated using TaqMan-based qPCR. Results Treatment of HUVEC with the pro-angiogenic stimuli VEGF, bFGF, HGF, or HB-EGF induced in transient phosphorylation (activation) of ATF2 at Thr69 and Thr71. Suppression of ATF2 function by overexpression of ATF2AA in endothelial cells strongly attenuated VEGF- and bFGF-induced tubular morphogenesis. Loss of ATF2 function resulted in a significant increase in the VEGF- and bFGF-induced upregulation of Notch ligands DLL1 and DLL4 suggesting ATF2 involvement in the upregulation of these proteins. Conclusion Our study demonstrates that growth factor-induced phosphorylation of ATF2 in residues 69 and 71 is essential for proper angiogenesis. Moreover, our results suggest that ATF2 plays a fundamental role in the regulation of Notch signalling-induced angiogenesis in endothelial cells. Conflict of interest None

Diabetes is associated with dysfunction of the neurovascular unit, although the mechanisms of this are incompletely understood and currently no treatment exists to prevent these negative effects. We previously found that the nitric oxide (NO) donor sodium nitroprusside (SNP) prevents the detrimental effect of glucose on neurovascular coupling in zebrafish. We therefore sought to establish the wider effects of glucose exposure on both the neurovascular unit and on behaviour in zebrafish, and the ability of SNP to prevent these. We incubated 4-days post-fertilisation (dpf) zebrafish embryos in 20 mM glucose or mannitol for 5 days until 9 dpf, with or without 0.1 mM SNP co-treatment for 24 h (8-9 dpf), and quantified vascular NO reactivity, vascular mural cell number, expression of a klf2a reporter, glial fibrillary acidic protein (GFAP) and transient receptor potential cation channel subfamily V member 4 (TRPV4), as well as spontaneous neuronal activation at 9 dpf, all in the optic tectum. We also assessed the effect on light/dark preference and locomotory characteristics during free-swimming studies. We find that glucose exposure significantly reduced NO reactivity, klf2a reporter expression, vascular mural cell number and TRPV4 expression, while significantly increasing spontaneous neuronal activation and GFAP expression (all in the optic tectum). Furthermore, when we examined larval behaviour, we found that glucose exposure significantly altered light/dark preference and high and low speed locomotion while in light. Co-treatment with SNP reversed all these molecular and behavioural effects of glucose exposure. Our findings comprehensively describe the negative effects of glucose exposure on the vascular anatomy, molecular phenotype and function of the optic tectum, and on whole-organism behaviour. We also show that SNP or other NO donors may represent a therapeutic strategy to ameliorate the complications of diabetes on the neurovascular unit. This article has an associated First Person interview with the first author of the paper.

Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-β and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4 , with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.

Endothelial cell behaviour during blood vessel formation is highly complex and dynamic. Transgenic zebrafish have provided many new insights into these processes, due to their ability to provide detailed in vivo imaging. We here report a previously undescribed endothelial cell behaviour during zebrafish embryonic development. Endothelial cells of the cerebral vessels of 3-5d post fertilisation embryos extruded large membranous spherical structures. These were only found on the cerebral vessels, and did not detach from the parent vessel, instead regressing back into the endothelial cell. These structures did not communicate with the vessel lumen, exhibited periodic oscillations in size and shape, and were enriched with filamentous actin at their neck. Due to their unknown nature and spherical appearance we termed these structures kugeln (German for sphere). Pharmacological inhibition of vascular endothelial growth factor (VEGF) signalling significantly increased kugel number while Notch inhibition significantly reduced both kugel number and diameter. Kugeln contain little cytoplasm, but are highly positive for nitric oxide (NO) reactivity, suggesting they represent a novel NO containing organelle specific to the cerebral vessels.

AbstractThe forkhead transcription factorsFoxc1andFoxc2are essential to establish intact vascular networks in mammals. How these genes interact with endothelial signalling pathways to exert their functions remains incompletely understood. We have generated novel zebrafish mutants infoxc1aandfoxc1b, the zebrafish orthologues of mammalianFoxc1, to determine their function during angiogenesis.foxc1amutants display abnormal formation of cranial veins including the primordial hindbrain channels (PHBC), reduced Vascular Endothelial Growth Factor (VEGF) receptor expression in these and loss of central arteries.foxc1bmutants are normal, whereasfoxc1a;foxc1bdouble mutants exhibit ectopic angiogenesis from trunk segmental arteries. Dll4/Notch signalling is reduced infoxc1a; foxc1bdouble mutant arteries and ectopic angiogenesis can be suppressed by induction of Notch or inhibition of Vegfc signalling. We conclude thatfoxc1aandfoxc1bplay compensatory and context-dependent roles to co-ordinate angiogenesis by promoting venous sprouting via induction of VEGF receptor expression whilst antagonising arterial sprouting by inducing Dll4/Notch signalling.foxc1a/bmediated induction of both pro- and anti-angiogenic axes of VEGF-Dll4/Notch negative feedback imparts competition to balance arterial and venous angiogenesis within developing vascular beds.Summary Statementfoxc1aandfoxc1bpromote angiogenesis from veins and suppress angiogenesis from arteries by promoting competing pro-angiogenic Vascular Endothelial Growth Factor signalling, and anti-angiogenic Dll4/Notch signalling in zebrafish embryos.

The forkhead transcription factors Foxc1 and Foxc2 are essential to establish intact vascular networks in mammals. How these genes interact with endothelial signalling pathways to exert their functions remains incompletely understood. We have generated novel zebrafish mutants in foxc1a and foxc1b , the zebrafish orthologues of mammalian Foxc1 , to determine their function during angiogenesis. foxc1a mutants display abnormal formation of cranial veins including the primordial hindbrain channels (PHBC), reduced Vascular Endothelial Growth Factor (VEGF) receptor expression in these and loss of central arteries. foxc1b mutants are normal, whereas foxc1a ; foxc1b double mutants exhibit ectopic angiogenesis from trunk segmental arteries. Dll4/Notch signalling is reduced in foxc1a; foxc1b double mutant arteries and ectopic angiogenesis can be suppressed by induction of Notch or inhibition of Vegfc signalling. We conclude that foxc1a and foxc1b play compensatory and context-dependent roles to co-ordinate angiogenesis by promoting venous sprouting via induction of VEGF receptor expression whilst antagonising arterial sprouting by inducing Dll4/Notch signalling. foxc1a / b mediated induction of both pro- and anti-angiogenic axes of VEGF-Dll4/Notch negative feedback imparts competition to balance arterial and venous angiogenesis within developing vascular beds.Summary Statement foxc1a and foxc1b promote angiogenesis from veins and suppress angiogenesis from arteries by promoting competing pro-angiogenic Vascular Endothelial Growth Factor signalling, and anti-angiogenic Dll4/Notch signalling in zebrafish embryos.

The transcriptional repressors Gfi1(a) and Gfi1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, Gfi1 and Gfi1b restrict or promote cell proliferation, prevent apoptosis, influence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation. In zebrafish, prRBCs express 2 of 3 zebrafish Gfi1/1b paralogs, Gfi1aa and Gfi1b. The recently identified zebrafish gfi1aa gene trap allele qmc551 drives erythroid green fluorescent protein (GFP) instead of Gfi1aa expression, yet homozygous carriers have normal prRBCs. prRBCs display a maturation defect only after splice morpholino-mediated knockdown of Gfi1b in gfi1aa qmc551 homozygous embryos. To study the transcriptome of the Gfi1aa/1b double-depleted cells, we performed an RNA-Seq experiment on GFP-positive prRBCs sorted from 20-hour-old embryos that were heterozygous or homozygous for gfi1aa qmc551 , as well as wt or morphant for gfi1b We subsequently confirmed and extended these data in whole-mount in situ hybridization experiments on newly generated single- and double-mutant embryos. Combined, the data showed that in the absence of Gfi1aa, the synchronously developing prRBCs were delayed in activating late erythroid differentiation, as they struggled to suppress early erythroid and endothelial transcription programs. The latter highlighted the bipotent nature of the progenitors from which prRBCs arise. In the absence of Gfi1aa, Gfi1b promoted erythroid differentiation as stepwise loss of wt gfi1b copies progressively delayed Gfi1aa-depleted prRBCs even further, showing that Gfi1aa and Gfi1b together set the pace for prRBC differentiation from hemangioblasts.

Protocol Summary Here we describe a method for high-throughput genotyping of live larval zebrafish as early as 72 h post-fertilization (hpf). Importantly, this technique allows rapid and cost-effective PCR-based genotyping from very small fin biopsies, which regenerate as the embryo develops, thereby allowing researchers to select embryos with desired genotypes to be raised to adulthood.