Molecular characterization of novel interferon gamma receptor 1 isoforms in zebrafish (Danio rerio) and goldfish (Carassius auratus L.)

Interferon gamma (IFNγ) is a highly pleotropic pro-inflammatory and anti-viral cytokine that mediates its effects by binding to a receptor complex composed of interferon gamma receptors 1 and 2 (IFNGR1 and IFNGR2). Using gene synteny analysis, we identified a distinct isoform of the zebrafish IFNGR1. The two zebrafish IFNGR1 called here IFNGR1-1 and IFNGR1-2 were used to identify the respective cDNA sequences of the goldfish IFNGR1-1 and IFNGR1-2. Analysis of protein sequences revealed that all fish IFNGR1 species have potential JAK1 and STAT1 docking sites. Phylogenetically, teleost IFNGR1 proteins grouped separately from those of higher vertebrates. Q-PCR analysis revealed that while the constitutive mRNA levels of the two zebrafish IFNGR1 isoforms were comparable in different tissues examined, the goldfish IFNGR1-1 tissue expression was substantially higher than that of IFNGR1-2. Q-PCR analysis of goldfish immune cell populations revealed highest expression of both receptor isoforms in monocytes. Incubation of goldfish macrophages with recombinant goldfish IFNγ2 (rgIFNγ2) up-regulated expression of both IFNGR1-1 and IFNGR1-2, while treatment of cells with rgTNFα2 only increased the expression of IFNGR1-1. Treatment with rgTGFβ resulted in more modest increases in expression of both receptor isoforms only after prolonged treatment. In vitro binding studies indicated that rgIFNGR1-1 bound to rgIFNγ1 but not rgIFNγ2, while the rgIFNGR1-2 bound to rgIFNγ2. Thus, unlike mammals that have a single IFNGR1, cyprinid fish have two distinct IFNGR1 isoforms that preferentially bind corresponding ligands, IFNγ1 and IFNγ2, respectively, suggesting that the type II interferon system of these fish species is distinct from that of higher vertebrates.     

Zebrafish ift57, ift88, and ift172 intraflagellar transport mutants disrupt cilia but do not affect hedgehog signaling

Cilia formation requires intraflagellar transport (IFT) proteins. Recent studies indicate that mammalian Hedgehog (Hh) signaling requires cilia. It is unclear, however, if the requirement for cilia and IFT proteins in Hh signaling represents a general rule for all vertebrates. Here we examine zebrafish ift57, ift88, and ift172 mutants and morphants for defects in Hh signaling. Although ift57 and ift88 mutants and morphants contained residual maternal protein, the cilia were disrupted. In contrast to previous genetic studies in mouse, mutations in zebrafish IFT genes did not affect the expression of Hh target genes in the neural tube and forebrain and had no quantitative effect on Hh target gene expression. Zebrafish IFT mutants also exhibited no dramatic changes in the craniofacial skeleton, somite formation, or motor neuron patterning. Thus, our data indicate the requirement for cilia in the Hh signal transduction pathway may not represent a universal mechanism in vertebrates. Developmental Dynamics 238:1744–1759, 2009. © 2009 Wiley‐Liss, Inc.     

The zebrafish as a model to study intestinal inflammation

Starting out as a model for developmental biology, during the last decade, zebrafish have also gained the attention of the immunologists and oncologists. Due to its small size, high fecundity and full annotation of its genome, the zebrafish is an attractive model system. The fact that fish are transparent early in life combined with the growing list of immune cell reporter fish, enables in vivo tracking of immune responses in a complete organism. Since zebrafish develop ex utero from a fertilized egg, immune development can be monitored from the start of life. Given that several gut functions and immune genes are conserved between zebrafish and mammals, the zebrafish is an interesting model organism to investigate fundamental processes underlying intestinal inflammation and injury. This review will first provide some background on zebrafish intestinal development, bacterial colonization and immunity, showing the similarities and differences compared to mammals. This will be followed by an overview of the existing models for intestinal disease, and concluded by future perspectives in light of the newest technologies and insights.     

Animal models of fibrodysplasia ossificans progressiva

Fibrodysplasia Ossificans Progressiva is a rare human disease of heterotopic ossification. FOP patients experience progressive development of ectopic bone within fibrous tissues that contributes to a gradual loss of mobility and can lead to early mortality. Due to lack of understanding of the etiology and progression of human FOP, and the fact that surgical interventions often exacerbate FOP disease progression, alternative therapeutic methods are needed, including modeling in animals, to study and improve understanding of human FOP. In this review we provide an overview of the existing animal models of FOP and the key mechanistic findings from each. In addition, we highlight the specific advantages of a new adult zebrafish model, generated by our lab, to study human FOP. Developmental Dynamics 247:279–288, 2018. © 2017 Wiley Periodicals, Inc.     

The neurotrophic properties of progranulin depend on the granulin E domain but do not require sortilin binding

Progranulin (PGRN) is a growth factor involved in wound healing, inflammation, tumor growth, and neurodegeneration. Mutations in the gene encoding PGRN give rise to shortage of PGRN and cause familial frontotemporal lobar degeneration. PGRN exerts neurotrophic functions and binding of PGRN to the membrane receptor sortilin (SORT1) mediates the endocytosis of PGRN. SORT1-mediated uptake plays an important role in the regulation of extracellular PGRN levels. We studied the role of SORT1 in PGRN-mediated neuroprotection in vitro and in vivo. The survival-enhancing effect of PGRN seemed to be dependent on the granulin E (GRN E) domain. Pharmacologic inhibition of the GRN E–SORT1 interaction or deletion of the SORT1 binding site of GRN E did not abolish its neurotrophic function. In addition, the in vivo phenotype of PGRN knockdown in zebrafish embryos was not phenocopied by SORT1 knockdown. These results suggest that GRN E mediates the neurotrophic properties of PGRN and that binding to SORT1 is not required for this effect.     

Evolution and function of chemokine receptors in the immune system of lower vertebrates

Chemokine receptors and their counterpart ligands are one of the evolutionary innovations of vertebrates. They play a guiding role in the coordination of cell trafficking in many biological processes. Comparative syntenic and phylogenetic analyses provide insight into the evolution of chemokine receptors and suggest that the repertoire of chemokine receptors varies in each species, regardless of the evolutionary position of the species. Despite the rapid evolution of chemokine receptors, the expression and function of orthologous chemokine receptors in lower and higher vertebrates are very similar. This is also true for the chemokine ligands that have been examined so far, such as CXCL8, CXCL12, and CCL25. As examples, this review will discuss how the evolution of the chemokine receptor CXCR4 is coincident with the emergence of lymphocytes in jawless vertebrates (lamprey); and that, in jawed vertebrates, CXCR4 and CCR9 are involved in thymus colonization. In myeloid cells, the function of CXCR1 in neutrophils and the expression of CXCR3 in macrophages and DCs are evolutionarily conserved between fish and mammals. In this context, medaka and zebrafish are outstanding models for studying the function of chemokines and their receptors.     

Understanding neurobehavioral genetics of zebrafish

Abstract

Due to its fully sequenced genome, high genetic homology to humans, external fertilization, fast development, transparency of embryos, low cost and active reproduction, the zebrafish (Danio rerio) has become a novel promising model organism in biomedicine. Zebrafish are a useful tool in genetic and neuroscience research, including linking various genetic mutations to brain mechanisms using forward and reverse genetics. These approaches have produced novel models of rare genetic CNS disorders and common brain illnesses, such as addiction, aggression, anxiety and depression. Genetically modified zebrafish also foster neuroanatomical studies, manipulating neural circuits and linking them to different behaviors. Here, we discuss recent advances in neurogenetics of zebrafish, and evaluate their unique strengths, inherent limitations and the rapidly growing potential for elucidating the conserved roles of genes in neuropsychiatric disorders.     

Expression of the Ku70 subunit (XRCC6) and protection from low dose ionizing radiation during zebrafish embryogenesis

The Ku70 protein, a product of the XRCC6 gene, is a component of the nonhomologous end-joining (NHEJ) pathway of DNA repair, which protects cells from the effects of radiation-induced DNA damage. Although the spatial expression of Ku70 during vertebrate embryogenesis has not been described, DNA repair proteins are generally considered to be "housekeeping" genes, which are required for radioprotection in all cells. Here, we report the cloning and characterization of the zebrafish Ku70 ortholog. In situ hybridization and RT-PCR analyses demonstrate that Ku70 mRNA is maternally provided and expressed uniformly among embryonic blastomeres. Later during embryogenesis, zygotically transcribed Ku70 mRNA specifically accumulates in neural tissue, including the retina and proliferative regions of the developing brain. In the absence of genotoxic stress, morpholino-mediated knockdown of Ku70 expression does not affect zebrafish embryogenesis. However, exposure of Ku70 morpholino-injected embryos to low doses of ionizing radiation leads to marked cell death throughout the developing brain, spinal cord, and tail. These results suggest that Ku70 protein plays a crucial role in protecting the developing nervous system from radiation-induced DNA damage during embryogenesis.     

Ethanol exposure alters zebrafish development: a novel model of fetal alcohol syndrome

Prenatal exposure to alcohol has been shown to produce the overt physical and behavioral symptoms known as fetal alcohol syndrome (FAS) in humans. Also, it is believed that low concentrations and/or short durations of alcohol exposure can produce more subtle effects. The purpose of this study was to investigate the effects of embryonic ethanol exposure on the zebrafish (Danio rerio) in order to determine whether this species is a viable animal model for studying FAS. Fertilized embryos were reared in varying concentrations of ethanol (1.5% and 2.9%) and exposure times (e.g., 0–8, 6–24, 12–24, and 48–72 h postfertilization; hpf); anatomical measures including eye diameter and heart rate were compared across groups. Results found that at the highest concentration of ethanol (2.9%), there were more abnormal physical distortions and significantly higher mortality rates than any other group. Embryos exposed to ethanol for a shorter duration period (0–8 hpf) at a concentration of 1.5% exhibited more subtle effects such as significantly smaller eye diameter and lower heart rate than controls. These results indicate that embryonic alcohol exposure affects external and internal physical development and that the severity of these effects is a function of both the amount of ethanol and the timing of ethanol exposure. Thus, the zebrafish represents a useful model for examining basic questions about the effects of embryonic exposure to ethanol on development.     

Zebrafish embryos express an orthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia

A wide range of drugs has been shown to prolong the QT interval of the electrocardiogram by blocking the pore-forming subunit of the rapidly activating delayed rectifier K+ channel, HERG (ether-à-go-go-related gene), sometimes leading to life-threatening arrhythmia. In this paper we describe cloning, sequence, and expression of the zebrafish orthologue of HERG, Zerg. Further, we studied effects of Zerg inhibition in zebrafish embryos caused by drugs or by an antisense approach. Zerg is expressed specifically in both heart chambers of zebrafish embryos, is composed of six transmembrane domains, and shows an especially high degree of amino acid conservation in the S6 and pore domain (99% identity). Several QT-prolonging drugs added to the bathing medium elicited bradycardia and arrhythmia in zebrafish embryos. The arrhythmia induced ranged from an atrioventricular 2:1 block, the ventricle beating half as often as the atrium, to more severe irregular arrhythmia with higher concentrations of the drugs. These effects were highly specific, reproducible, and rapid, e.g., 10 microM astemizole caused a 2:1 heartbeat within a minute after addition of the compound in all the embryos studied. Morpholino antisense oligonucleotides targeting Zerg were injected into zebrafish embryos and elicited similar dose-sensitive and specific arrhythmia as the QT-prolonging drugs, suggesting an evolutionarily conserved role for Erg in regulating heartbeat rate and rhythm. Further, we identified a mutation in the Per-Arnt-Sim domain of the Zerg channel in the breakdance mutant, also characterized by a 2:1 atrioventricular block. In conclusion, the zebrafish could be a tractable model organism for the study of Erg function and modulation but might also have a value in the field of cardiovascular pharmacology, e.g., as an early preclinical model for testing drugs under development for potential QT prolongation.