The oxygen-sensing transcription factor hypoxia-inducible factor-1␣ (HIF-1␣) plays a critical role in the regulation of myeloid cell function. The mechanisms of regulation are not well understood, nor are the phenotypic consequences of HIF modulation in the context of neutrophilic inflammation. Species conservation across higher metazoans enables the use of the genetically tractable and transparent zebrafish (Danio rerio) embryo to study in vivo resolution of the inflammatory response. Using both a pharmacologic approach known to lead to stabilization of HIF-1␣, and selective genetic manipulation of zebrafish HIF-1␣ homologs, we sought to determine the roles of HIF-1␣ in inflammation resolution. Both approaches reveal that activated Hif-1␣ delays resolution of inflammation after tail transection in zebrafish larvae. This delay can be replicated by neutrophil-specific Hif activation and is a consequence of both reduced neutrophil apoptosis and increased retention of neutrophils at the site of tissue injury. Hif-activated neutrophils continue to patrol the injury site during the resolution phase, when neutrophils would normally migrate away. Sitedirected mutagenesis of Hif in vivo reveals that hydroxylation of Hif-1␣ by prolyl hydroxylases critically regulates the Hif pathway in zebrafish neutrophils. Our data demonstrate that Hif-1␣ regulates neutrophil function in complex ways during inflammation resolution in vivo. (Blood. 2011;118(3):712-722) IntroductionNeutrophilic inflammation is of fundamental importance in the innate immune response to bacterial and fungal infection in vertebrates, and it can be initiated by sterile tissue injury. Irrespective of its etiology, inflammation must resolve in a timely manner to avoid damage to surrounding tissues. 1 Persisting, noninfectious inflammation is the hallmark of inflammatory diseases, a major cause of morbidity and mortality in the developed world. The resolution phase of inflammation is critical to the restoration of normal tissue function after an inflammatory response, and thus has a central role in determining the outcome of inflammation. 2 Despite the central place of failed resolution in the pathogenesis of inflammatory disease, much remains to be known about the cellular and molecular events involved.Although neutrophil apoptosis, and subsequent uptake and removal by macrophages (efferocytosis), is well documented as a disposal route for inflammatory neutrophils, [3][4][5] there is emerging evidence that other mechanisms also may contribute to certain types of inflammation resolution. In the lung, some neutrophils are lost into the airways and expectorated, 6 and in rheumatoid arthritis, neutrophils may leave the inflammatory site while still alive and re-enter the circulation. 7 Neutrophils also can be removed by migration through tissues away from the infection site; a process termed retrograde chemotaxis, or reverse migration. [8][9][10] This process is most widely characterized in the zebrafish (Danio rerio) embryo and is less well studied in mammalian s...

Tuberculosis is a current major world-health problem, exacerbated by the causative pathogen, Mycobacterium tuberculosis (Mtb), becoming increasingly resistant to conventional antibiotic treatment. Mtb is able to counteract the bactericidal mechanisms of leukocytes to survive intracellularly and develop a niche permissive for proliferation and dissemination. Understanding of the pathogenesis of mycobacterial infections such as tuberculosis (TB) remains limited, especially for early infection and for reactivation of latent infection. Signaling via hypoxia inducible factor α (HIF-α) transcription factors has previously been implicated in leukocyte activation and host defence. We have previously shown that hypoxic signaling via stabilization of Hif-1α prolongs the functionality of leukocytes in the innate immune response to injury. We sought to manipulate Hif-α signaling in a well-established Mycobacterium marinum (Mm) zebrafish model of TB to investigate effects on the host's ability to combat mycobacterial infection. Stabilization of host Hif-1α, both pharmacologically and genetically, at early stages of Mm infection was able to reduce the bacterial burden of infected larvae. Increasing Hif-1α signaling enhanced levels of reactive nitrogen species (RNS) in neutrophils prior to infection and was able to reduce larval mycobacterial burden. Conversely, decreasing Hif-2α signaling enhanced RNS levels and reduced bacterial burden, demonstrating that Hif-1α and Hif-2α have opposing effects on host susceptibility to mycobacterial infection. The antimicrobial effect of Hif-1α stabilization, and Hif-2α reduction, were demonstrated to be dependent on inducible nitric oxide synthase (iNOS) signaling at early stages of infection. Our findings indicate that induction of leukocyte iNOS by stabilizing Hif-1α, or reducing Hif-2α, aids the host during early stages of Mm infection. Stabilization of Hif-1α therefore represents a potential target for therapeutic intervention against tuberculosis.

Key Points Neutrophil lifespan is extended in patients with gain-of-function HIF2A mutations. HIF-2α regulates in vivo neutrophil longevity and thus tissue inflammation and repair.

We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe hyperventilation and cardiophysiologic response. The vhl mutants develop polycythemia with concomitantly increased epo/epor mRNA levels and erythropoietin signaling. In situ hybridizations reveal global up-regulation of both red and white hematopoietic lin-

Background: Aberrant activation of the Hedgehog (Hh) signaling pathway in different organisms has shown the importance of this family of morphogens during development. Genetic screens in zebrafish have assigned specific roles for Hh in proliferation, differentiation and patterning, but mainly as a result of a loss of its activity. We attempted to fully activate the Hh pathway by removing both receptors for the Hh proteins, called Patched1 and 2, which are functioning as negative regulators in this pathway.

In a large-scale screen, we isolated mutants displaying a specific visible phenotype in embryos or early larvae of the zebrafish, Danio rerio. Males were mutagenized with ethylnitrosourea (ENU) and F2 families of single pair matings between sibling F1 fish, heterozygous for a mutagenized genome, were raised. Egg lays were obtained from several crosses between F2 siblings, resulting in scoring of 3857 mutagenized genomes. F3 progeny were scored at the second, third and sixth day of development, using a stereomicroscope. In a subsequent screen, fixed embryos were analyzed for correct retinotectal projection. A total of 4264 mutants were identified. Two thirds of the mutants displaying rather general abnormalities were eventually discarded. We kept and characterized 1163 mutants. In complementation crosses performed between mutants with similar phenotypes, 894 mutants have been assigned to 372 genes. The average allele frequency is 2.4. We identified genes involved in early development, notochord, brain, spinal cord, somites, muscles, heart, circulation, blood, skin, fin, eye, otic vesicle, jaw and branchial arches, pigment pattern, pigment formation, gut, liver, motility and touch response. Our collection contains alleles of almost all previously described zebrafish mutants. From the allele frequencies and other considerations we estimate that the 372 genes defined by the mutants probably represent more than half of all genes that could have been discovered using the criteria of our screen. Here we give an overview of the spectrum of mutant phenotypes obtained, and discuss the limits and the potentials of a genetic saturation screen in the zebrafish.

SUMMARYBiallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the development of highly vascularized neoplasms in multiple organ systems. We show that zebrafish vhl mutants display a marked increase in blood vessel formation throughout the embryo, starting at 2 days post-fertilization. The most severe neovascularization is observed in distinct areas that overlap with high vegfa mRNA expression, including the vhl mutant brain and eye. Real-time quantitative PCR revealed increased expression of the duplicated VEGFA orthologs vegfaa and vegfab, and of vegfb and its receptors flt1, kdr and kdr-like, indicating increased vascular endothelial growth factor (Vegf) signaling in vhl mutants. Similar to VHL-associated retinal neoplasms, diabetic retinopathy and age-related macular degeneration, we show, by tetramethyl rhodaminedextran angiography, that vascular abnormalities in the vhl -/-retina lead to vascular leakage, severe macular edema and retinal detachment. Significantly, vessels in the brain and eye express cxcr4a, a marker gene expressed by tumor and vascular cells in VHL-associated hemangioblastomas and renal cell carcinomas. VEGF receptor (VEGFR) tyrosine kinase inhibition (through exposure to sunitinib and 676475) blocked vhl -/--induced angiogenesis in all affected tissues, demonstrating that Vegfaa, Vegfab and Vegfb are key effectors of the vhl -/-angiogenic phenotype through Flt1, Kdr and Kdr-like signaling. Since we show that the vhl -/-angiogenic phenotype shares distinct characteristics with VHL-associated vascular neoplasms, zebrafish vhl mutants provide a valuable in vivo vertebrate model to elucidate underlying mechanisms contributing to the development of these lesions. Furthermore, vhl mutant zebrafish embryos carrying blood vessel-specific transgenes represent a unique and clinically relevant model for tissue-specific, hypoxia-induced pathological angiogenesis and vascular retinopathies. Importantly, they will allow for a cost-effective, non-invasive and efficient way to screen for novel pharmacological agents and combinatorial treatments. Yancopoulos et al., 2000;Carmeliet, 2005). Recently, it was shown that autocrine VEGFA signaling is also required for vascular homeostasis (Lee et al., 2007). VEGFA acts through two high affinity receptor tyrosine kinases, FLT-1 (VEGFR1) and . KDR is thought to mediate most of the angiogenic functions of VEGFA, whereas FLT1 may act as a sink for VEGFA, or as a decoy receptor to negatively regulate signaling through KDR (Yancopoulos et al., 2000). Disease Models & Mechanisms DMMOverproduction of hypoxia-inducible mRNAs, including VEGFA, is a hallmark of the highly vascularized neoplasms associated with biallelic inactivation of the VHL tumor suppressor gene, including hemangioblastomas of the retina and central nervous system (CNS), and clear cell renal cell carcinoma (ccRCC) (Maher et al., 1991;Maher and Kaelin, 1997;Van Poppel et al., 2000;Lonser et al., 2003). Hemangioblastomas are the most common manifestati...

A low level of tissue oxygen (hypoxia) is a physiological feature of a wide range of diseases, from cancer to infection. Cellular hypoxia is sensed by oxygen-sensitive hydroxylase enzymes, which regulate the protein stability of hypoxia-inducible factor α (HIF-α) transcription factors. When stabilised, HIF-α binds with its cofactors to HIF-responsive elements (HREs) in the promoters of target genes to coordinate a wide-ranging transcriptional programme in response to the hypoxic environment. This year marks the 20th anniversary of the discovery of the HIF-1α transcription factor, and in recent years the HIF-mediated hypoxia response is being increasingly recognised as an important process in determining the outcome of diseases such as cancer, inflammatory disease and bacterial infections. Animal models have shed light on the roles of HIF in disease and have uncovered intricate control mechanisms that involve multiple cell types, observations that might have been missed in simpler in vitro systems. These findings highlight the need for new whole-organism models of disease to elucidate these complex regulatory mechanisms. In this Review, we discuss recent advances in our understanding of hypoxia and HIFs in disease that have emerged from studies of zebrafish disease models. Findings from such models identify HIF as an integral player in the disease processes. They also highlight HIF pathway components and their targets as potential therapeutic targets against conditions that range from cancers to infectious disease.