Removal of ribonucleotides from DNA by RNaseH2 is essential for genome stability, and its impacted function causes the neurodegenerative disease, Aicardi Goutières Syndrome. We have created a zebrafish rnaseh2a mutant to model this process. Surprisingly, RNaseH2a knockouts show little phenotypic abnormality at adulthood in the first generation, unlike mouse knockout models, which are early embryonic lethal. However, the second generation offspring show reduced development, increased ribonucleotide incorporation and upregulation of key inflammatory markers, resulting in both maternal and paternal embryonic lethality. Thus, neither fathers or mothers can generate viable offspring even when crossed to wild-type partners. Despite their survival, rnaseh2a-/- adults show an accumulation of ribonucleotides in both the brain and testes that is not present in early development. Our data suggest that homozygotes possess RNaseH2 independent compensatory mechanisms that are inactive or overwhelmed by the inherited ribonucleotides in their offspring, or that zebrafish have a yet unknown tolerance mechanism. Additionally, we identify ribodysgenesis, the rapid removal of rNMPs and subsequently lethal fragmentation of DNA as responsible for maternal and paternal embryonic lethality.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal failure in humans and results from germline mutations in PKD1 or PKD2 . Despite the recent approval of tolvaptan, safer and more effective alternative drugs are clearly needed to slow disease progression. As a first step in drug discovery, we conducted an unbiased chemical screen on zebrafish pkd2 mutant embryos using two publicly available compound libraries (Spectrum, PKIS) totalling 2,367 compounds to identify novel treatments for ADPKD. Using dorsal tail curvature as the assay readout, three major chemical classes (steroids, coumarins, flavonoids) were identified from the Spectrum library as the most promising candidates to be tested on human PKD1 cystic cells. Amongst these were an androgen, 5α−androstane 3,17-dione, detected as the strongest enhancer of the pkd2 phenotype but whose effect was found to be independent of the canonical androgen receptor pathway. From the PKIS library, we identified several ALK5 kinase inhibitors as strong suppressors of the pkd2 tail phenotype and in vitro cyst expansion. In summary, our results identify ALK5 and non-canonical androgen receptors as potential therapeutic targets for further evaluation in drug development for ADPKD.

Oxygen is a central molecule in the development of multicellular life, allowing efficient energy generation. Inadequate oxygen supply requires rapid adaptations to prevent cellular damage and the hypoxia-inducible factor (HIF) pathway plays a central role in this adaptation. Numerous diseases and disease processes are influenced by hypoxia and the HIF pathway. One component, von Hippel Lindau (VHL), is a well-known tumor suppressor, which acts at least in part via regulating HIF signaling. The zebrafish has become a central vertebrate model organism in which developmental and disease processes can be studied. In this review, we have tried to bring together knowledge on the HIF/ hypoxic signaling pathway in zebrafish, including what is known on VHL functions.

Nitric oxide (NO) is a gaseous neurotransmitter that has important behavioural functions in the vertebrate brain. In this study we compare the impact of decreased nitric NO signalling upon behaviour and neurobiology using both zebrafish and mouse. nitric oxide synthase mutant (nos1−/−) zebrafish show significantly reduced aggression and an increase in anxiety-like behaviour without altered production of the stress hormone cortisol. Nos1−/− mice also exhibit decreased aggression and are hyperactive in an open field test. Upon reduction of NO signalling, monoamine neurotransmitter metabolism is reduced as a consequence of decreased Monoamine oxidase activity. Treatment of nos1−/− zebrafish with the 5-HT receptor 1A agonist 8-OH-DPAT rescues aggression and some aspects of anxiety-like behaviour. Taken together, the interplay between NO and 5-HT appears to be critical to control behaviour. Our cross-species approach challenges the previous notion that reduced neuronal NOS leads to increased aggression. Rather, Nos1 knock-out can also lead to decreased aggression in some situations, a finding that may have implications for future translational research.

The goal of personalised medicine is to develop tailor-made therapies for patients in whom currently available therapeutics fail. This approach requires correlating individual patient genotype data to specific disease phenotype data and using these stratified data sets to identify bespoke therapeutics. Applications for personalised medicine include common complex diseases which may have multiple targets, as well as rare monogenic disorders, for which the target may be unknown. In both cases, whole genome sequence analysis (WGS) is discovering large numbers of disease associated mutations in new candidate genes and potential modifier genes. Currently, the main limiting factor is the determination of which mutated genes are important for disease progression and therefore represent potential targets for drug discovery. Zebrafish have gained popularity as a model organism for understanding developmental processes, disease mechanisms and more recently for drug discovery and toxicity testing. In this chapter, we will examine the diverse roles that zebrafish can make in the expanding field of personalised medicine, from generating humanised disease models to xenograft screening of different cancer cell lines, through to finding new drugs via in vivo phenotypic screens. We will discuss the tools available for zebrafish research and recent advances in techniques, highlighting the advantages and potential of using zebrafish for high throughput disease modeling and precision drug discovery.

Introduction: The zebrafish is an excellent model for imaging vascular development owing to its optical clarity and the ability to generate tissue-specific transgenic reporter lines, which have been previously used to indicate cell location and shape. More sophisticated transgenics can provide subcellular signalling data. Since calcium signalling is known to lie downstream of VEGF during angiogenesis, we have generated the novel fli1a-gff:uas-GCaMP7a transgenic line allowing real-time imaging and dissection of the contribution of calcium signalling during this process. We have also generated the novel fli1a:lifeact-clover transgenic to visualise fliopodial dynamics during angiogenesis and a fli1a:parvalbumin-α-V2AmCherry construct to conditionally inhibit calcium signalling in endothelial cells (ECs). By employing these tools in combination with high-resolution light sheet fluorescence microscopy (LSFM), we can visualise endothelial biology in a manner not previously possible in vivo.

Study rationale: Hypoxic signalling is a potent angiogenic stimulus in vivo. Mutation of the von Hippel Lindau (vhl) gene in zebrafish activates hypoxia signalling, resulting in excessive and aberrant angiogenesis during development. Tissue macrophages have been shown to enhance anastomotic fusion of endothelial angiogenic sprouts. We hypothesised that macrophages may contribute to the aberrant angiogenesis seen in vhl mutants.

Neutrophilic inflammation plays an important role in inflammatory lung diseases but therapeutic targeting of neutrophil (PMN) persistence is lacking. PMN lifespan and function is regulated by hypoxia, a characteristic feature of inflamed tissues, via the HIF/VHL/hydroxylase pathway, specifically hypoxia inducible factor-1α (HIF-1α) and prolyl hydroxylase-3 (PHD3). Targeting HIF-1α in myeloid cells impaired immune function, but PHD3 regulated PMN lifespan without affecting function. Given that PHD3 preferentially regulates HIF-2α, we investigated the role of HIF-2α in PMN-mediated inflammation. Peripheral blood PMNs isolated from healthy volunteers and mice expressed HIF-2α and expression was enhanced by heat-killed bacteria. Using PMNs isolated from patients with active inflammatory arthritis (IA) we demonstrated significant upregulation of HIF2A mRNA (IA 92.9±30.3 vs. control 4.3±0.9 AU relative to ACTB, P<0.05) and protein (IA 0.26±0.05 vs. control 0.01±0.01 OD relative to P38, P<0.01) in circulating inflammatory PMNs. PMNs recruited to the airways of patients with COPD also displayed strong HIF-2α staining. The consequences of HIF-2α upregulation were examined using human PMNs from patients with gain-of-function mutations in the HIF2A gene. Neutrophils isolated from these patients had reduced rates of constitutive apoptosis. Recapitulation of the human HIF2A mutations in the orthologous HIF2A gene, epas1a, in zebrafish delayed resolution of inflammation in a tail injury model (24 hrs post injury, epas1a 12.7±1.4 vs. ctrl 5.2±0.5 PMNs, p<0.001) with an associated reduction in PMN apoptosis (epas1a 1.0% vs. ctrl 1.6%, p<0.05). Mice with myeloid-specific deletion of Hif2a had normal PMN survival in response to hypoxia and the cells showed no functional defect in vitro. Importantly, in a PMN-mediated acute lung injury model, myeloid-specific deficiency of HIF-2α markedly enhanced resolution of inflammation (BAL PMN count 48 hours following nebulised LPS, WT 2.13×106±0.08 vs. KO 1.39×106±0.24, p<0.05) and reduced lung injury (BAL IgM at 48 hours, WT 211±22.8 vs. KO 74.7±27.2 ng/ml), implicating HIF-2α in PMN persistence in inflamed lung tissue. These data support a critical and selective role for HIF-2α in the resolution of inflammation through the maintenance of PMN survival, and provide a platform to dissect the therapeutic utility of targeting HIF-2α in chronic inflammatory diseases.

Introduction Hypoxia drives angiogenesis in a range of pathologies. Mutations in von hippel lindau protein (vhl) lead to excessive angiogenesis via upregulation of hypoxic signalling, due to impaired HIF-1α degradation. Physical forces exerted by blood flow have been shown to contribute to vascular remodelling. We therefore used vhl mutant zebrafish to observe the interplay between hypoxic signalling, haemodynamic flow and vascular development. Since NO has been shown to be both pro-angiogenic and released in response to haemodynamic force, we assessed whether NO contributed to angiogenesis in this model. Methods Vhl mutant zebrafish were crossed with a fli1; GFP transgenic that expresses Green Fluorescent Protein (GFP) in the endothelium. Embryonic vascular development was observed in mutants and wild type siblings by confocal microscopy. To determine the role of blood flow in the angiogenic response, cardiac troponin t2 was knocked down by morpholino antisense injection. To assess the contribution of nitric oxide, embryos were treated with either L-NAME (nitric oxide synthase inhibitor) (1mM) or sodium nitroprusside (NO donor) (100μM) from 24-h post fertilisation (hpf) until imaging at 4dpf. Results Imaging of the developing trunk vasculature revealed that vhl mutant embryos display excessive and aberrant angiogenesis from 3dpf (Abstract 72 figure 1A, B). Cardiac troponin T2 knockdown prevented any cardiac contraction, but embryos develop normally due to passive oxygen diffusion. Loss of blood flow did not alter normal intersegmental vessel patterning in either controls (Abstract 72 figure 1C) or vhl mutants (Abstract 72 figure 1D). However, loss of blood flow completely prevented excessive angiogenesis in vhl mutants (Abstract 72 figures 1D and 2), implying that both blood flow and hypoxic signalling are required for “pathological” angiogenesis but not developmental angiogenesis (vasculogenesis). NO synthase inhibition with L-NAME had no effect, suggesting that the contribution of flow to excessive angiogenesis in response to upregulated hypoxic signalling is NO independent. Conclusion Angiogenesis in response to hypoxic signalling is critically dependent upon haemodynamic force, compared with developmental vasculogenesis that can proceed in the absence of any blood flow. This indicates a different mechanism of development for hypoxia driven angiogenesis and vasculogenesis which may have important therapeutic implications.

Rationale Mutations in the PKD2 gene cause human autosomal dominant polycystic kidney disease (ADPKD). ADPKD is associated with vascular complications independent of renal dysfunction but little is known about the role of PKD2 in vascular development. We therefore characterised vascular development in PKD2 mutant zebrafish. Methodology We identified a novel PKD2 mutant zebrafish generated in a previously unpublished N-ethyl-N-nitrosourea mutagenesis screen. This was crossed with endothelial transgenic lines fli1:EGFP (cytoplasmic GFP) and flk1:nlsEGFP (nuclear localised GFP). Serial confocal microscopy was used to image the developing vasculature of PKD2 mutants and wildtype siblings until 5-day old. Mutant embryos were stained with phalloidin that labels muscle cells. Results Genotyping revealed the PKD2 mutation to be g.5860G>A, leading to a premature stop codon in exon 5. As described for other zebrafish PKD2 mutant alleles, homozygous mutants had a pronounced dorsal curvature but otherwise developed normally with no delay in onset of circulation. When we quantified endothelial number in the intersomitic vessels (ISVs) running between the somites we found this was almost doubled in PKD2 mutants by 3-day old (p<0.001) and persisted until at least 5-day old. When we characterised ISV morphology, we observed highly abnormal loops that were never observed in wildtypes. Phalloidin staining showed no alteration in somite structure to account for abnormal ISV patterning. Conclusion This is the first description of hyperproliferative angiogenesis induced by mutations in PKD2. The mechanism needs to be further delineated but our data suggest abnormal vessel development may underlie some vascular complications of ADPKD.

Cilia perform essential motile and sensory functions central to many developmental and physiological processes. Disruption of their structure or function can have profound phenotypic consequences, and has been linked to left-right patterning and polycystic kidney disease. In a forward genetic screen for mutations affecting ciliary motility, we isolated zebrafish mutant hu255H. The mutation was found to disrupt an ortholog of the uncharacterized highly conserved human SDS22-like leucine-rich repeat(LRR)-containing protein LRRC50 (16q24.1) and Chlamydomonas Oda7p. Zebrafish lrrc50 is specifically expressed in all ciliated tissues. lrrc50(hu255H) mutants develop pronephric cysts with an increased proliferative index, severely reduced brush border, and disorganized pronephric cilia manifesting impaired localized fluid flow consistent with ciliary dysfunction. Electron microscopy analysis revealed ultrastructural irregularities of the dynein arms and misalignments of the outer-doublet microtubules on the ciliary axonemes, suggesting instability of the ciliary architecture in lrrc50(hu255H) mutants. TheSDS22-like leucine-rich repeats present in Lrrc50 are necessary for proper protein function, since injection of a deletion construct of the first LRR did not rescue the zebrafish mutant phenotype. Subcellular distribution of human LRRC50-EGFP in MDCK and HEK293T cells is diffusely cytoplasmic and concentrated at the mitotic spindle poles and cilium. LRRC50 RNAi knock-down in human proximal tubule HK-2 cells thoroughly recapitulated the zebrafish brush border and cilia phenotype, suggesting conservation of LRRC50 function between both species. In summary, we present the first genetic vertebrate model for lrrc50 function and propose LRRC50 to be a novel candidate gene for human cystic kidney disease, involved in regulation of microtubule-based cilia and actin-based brush border microvilli.