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Aims: Nitrite is reduced to nitric oxide (NO) under physiological and pathological hypoxic conditions to modulate angiogenesis and improve ischemia–reperfusion injury. Although adult mammals lack the ability to regenerate the heart after injury, this is preserved in neonates and efforts to reactivate this process are of great interest. Unlike mammals, the adult zebrafish maintain the innate ability to regenerate their hearts after injury, providing an important model to study cardiac regeneration. We thus explored the effects of physiological levels of nitrite on cardiac and fin regeneration and downstream cellular and molecular signaling pathways in response to amputation and cryoinjury. Results: Nitrite treatment of zebrafish after ventricular amputation or cryoinjury to the heart in hypoxic water (∼3 parts per million of oxygen) increases cardiomyocyte proliferation, improves angiogenesis, and enhances early recruitment of thrombocytes, macrophages, and neutrophils to the injury. When tested in a fin regeneration model, neutrophil recruitment to the injury site was found to be dependent on NO. Innovation: This is the first study to evaluate effects of physiological levels of nitrite on cardiac regeneration in response to cardiac injury, with the observation that nitrite in water accelerates zebrafish heart regeneration. Conclusion: Physiological and therapeutic levels of nitrite increase thrombocyte, neutrophil, and macrophage recruitment to the heart after amputation and cryoinjury in zebrafish, resulting in accelerated cardiomyocyte proliferation and angiogenesis. Translation of this finding to mammalian models of injury during early development may provide an opportunity to improve outcomes during intrauterine fetal or neonatal cardiac surgery.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal-dominant vascular disorder characterized by development of high-flow arteriovenous malformations (AVMs) that can lead to stroke or high-output heart failure. HHT2 is caused by heterozygous mutations in ACVRL1, which encodes an endothelial cell bone morphogenetic protein (BMP) receptor, ALK1. BMP9 and BMP10 are established ALK1 ligands. However, the unique and overlapping roles of these ligands remain poorly understood. To define the physiologically relevant ALK1 ligand(s) required for vascular development and maintenance, we generated zebrafish harboring mutations in bmp9 and duplicate BMP10 paralogs, bmp10 and bmp10-like. bmp9 mutants survive to adulthood with no overt phenotype. In contrast, combined loss of bmp10 and bmp10-like results in embryonic lethal cranial AVMs indistinguishable from acvrl1 mutants. However, despite embryonic functional redundancy of bmp10 and bmp10-like, bmp10 encodes the only required Alk1 ligand in the juvenile-to-adult period. bmp10 mutants exhibit blood vessel abnormalities in anterior skin and liver, heart dysmorphology, and premature death, and vascular defects correlate with increased cardiac output. Together, our findings support a unique role for Bmp10 as a non-redundant Alk1 ligand required to maintain the post-embryonic vasculature and establish zebrafish bmp10 mutants as a model for AVM-associated high-output heart failure, which is an increasingly recognized complication of severe liver involvement in HHT2.

Since the discovery of new members of the globin superfamily such as Cytoglobin, Neuroglobin and Globin X, in addition to the most well-known members, Hemoglobin and Myoglobin, different hypotheses have been suggested about their function in vertebrates. Globins are ubiquitously found in living organisms and can carry out different functions based on their ability to bind ligands such as O2, and nitric oxide (NO) and to catalyze reactions scavenging NO or generating NO by reducing nitrite. NO is a highly diffusible molecule with a central role in signaling important for egg maturation, fertilization and early embryonic development. The globins ability to scavenge or generate NO makes these proteins ideal candidates in regulating NO homeostasis depending on the micro environment and tissue NO demands. Different amounts of various globins have been found in zebrafish eggs and developing embryos where it's unlikely that they function as respiratory proteins and instead could play a role in maintaining embryonic NO homeostasis. Here we summarize the current knowledge concerning the role of NO in adult fish in comparison to mammals and we discuss NO function during embryonic development with possible implications for globins in maintaining embryonic NO homeostasis.

Aims Notch and activin receptor-like kinase 1 (ALK1) have been implicated in arterial specification, angiogenic tip/stalk cell differentiation, and development of arteriovenous malformations (AVMs), and ALK1 can cooperate with Notch to up-regulate expression of Notch target genes in cultured endothelial cells. These findings suggest that Notch and ALK1 might collaboratively program arterial identity and prevent AVMs. We therefore sought to investigate the interaction between Notch and Alk1 signalling in the developing vertebrate vasculature. Methods and results We modulated Notch and Alk1 activities in zebrafish embryos and examined effects on Notch target gene expression and vascular morphology. Although Alk1 is not necessary for expression of Notch target genes in arterial endothelium, loss of Notch signalling unmasks a role for Alk1 in supporting hey2 and ephrinb2a expression in the dorsal aorta. In contrast, Notch and Alk1 play opposing roles in hey2 expression in cranial arteries and dll4 expression in all arterial endothelium, with Notch inducing and Alk1 repressing these genes. Although alk1 loss increases expression of dll4 , AVMs in alk1 mutants could neither be phenocopied by Notch activation nor rescued by Dll4/Notch inhibition. Conclusion Control of Notch targets in arterial endothelium is context-dependent, with gene-specific and region-specific requirements for Notch and Alk1. Alk1 is not required for arterial identity, and perturbations in Notch signalling cannot account for alk1 mutant-associated AVMs. These data suggest that AVMs associated with ALK1 mutation are not caused by defective arterial specification or altered Notch signalling.

The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51±10.35 μm) in opposition to blood flow (i.e. subtending 142.170±21.170 with the flow direction).

Heterozygous loss of the arterial-specific TGF-β type I receptor, activin receptor-like kinase 1 (ALK1), causes hereditary hemorrhagic telangiectasia (HHT). HHT is characterized by development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). However, how decreased ALK1 signaling leads to AVMs is unknown. To understand the cellular missteps that cause AVMs, we assessed endothelial cell behavior in alk1-deficient zebrafish embryos, which develop cranial AVMs. Our data demonstrate that alk1 loss has no effect on arterial endothelial cell proliferation but alters arterial endothelial cell migration within lumenized vessels. In wild type embryos, alk1-positive cranial arterial endothelial cells generally migrate toward the heart, against the direction of blood flow, with some cells incorporating into endocardium. In alk1-deficient embryos, migration against flow is dampened and migration in the direction of flow is enhanced. Altered migration results in decreased endothelial cell number in arterial segments proximal to the heart and increased endothelial cell number in arterial segments distal to the heart. We speculate that the consequent increase in distal arterial caliber and hemodynamic load precipitates the flow-dependent development of downstream AVMs.