Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning

Caveolae are an abundant feature of many animal cells. However, the exact function of caveolae remains unclear. We have used the zebrafish, Danio rerio, as a system to understand caveolae function focusing on the muscle-specific caveolar protein, caveolin-3 (Cav3). We have identified caveolin-1 (alpha and beta), caveolin-2 and Cav3 in the zebrafish. Zebrafish Cav3 has 72% identity to human CAV3, and the amino acids altered in human muscle diseases are conserved in the zebrafish protein. During embryonic development, cav3 expression is apparent by early segmentation stages in the first differentiating muscle precursors, the adaxial cells and slightly later in the notochord. cav3 expression appears in the somites during mid-segmentation stages and then later in the pectoral fins and facial muscles. Cav3 and caveolae are located along the entire sarcolemma of late stage embryonic muscle fibers, whereas beta-dystroglycan is restricted to the muscle fiber ends. Down-regulation of Cav3 expression causes gross muscle abnormalities and uncoordinated movement. Ultrastructural analysis of isolated muscle fibers reveals defects in myoblast fusion and disorganized myofibril and membrane systems. Expression of the zebrafish equivalent to a human muscular dystrophy mutant, CAV3P104L, causes severe disruption of muscle differentiation. In addition, knockdown of Cav3 resulted in a dramatic up-regulation of eng1a expression resulting in an increase in the number of muscle pioneer-like cells adjacent to the notochord. These studies provide new insights into the role of Cav3 in muscle development and demonstrate its requirement for correct intracellular organization and myoblast fusion.

Zebrafish KLF4 is essential for anterior mesendoderm/pre-polster differentiation and hatching

Gene knockout studies of Kruppel-like factors (KLFs) in mice have shown essential roles in organogenesis. A screen for KLF family members in zebrafish identified many KLFs. One of these, zebrafish KLF4 (zKLF4) is the homologue of neptune, a Xenopus laevis KLF. zKLF4 is expressed from approximately 80% epiboly a patch of dorsal/anterior mesendodermal cells called the pre-polster and, subsequently, in the polster and hatching gland. Here we investigate the function of zKLF4 using morpholino-based antisense oligonucleotides. Knockdown of zKLF4 resulted in complete absence of hatching gland formation and subsequent hatching in zebrafish. In addition, there was early knockdown of expression of the pre-polster/anterior mesendoderm markers CatL, cap1, and BMP4. These results indicate zKLF4 is expressed within the pre-polster, an early mesendodermal site, and that it plays a critical role in the differentiation of these cells into hatching gland cells.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development

The Crim1 gene encodes a transmembrane protein containing six cysteine-rich repeats similar to those found in the BMP antagonist, chordin (chd). To investigate its physiological role, zebrafish crim1 was cloned and shown to be both maternally and zygotically expressed during zebrafish development in sites including the vasculature, intermediate cell mass. notochord, and otic vesicle. Bent or hooked tails with U-shaped somites were observed in 85% of morphants from 12 hpf. This was accompanied by a loss of muscle pioneer cells. While morpholino knockdown of crim1 showed some evidence of ventralisation, including expansion of the intermediate cell mass (ICM), reduction in head size bent tails and disruption to the somites and notochord, this did not mimic the classically ventralised phenotype, as assessed by the pattern of expression of the dorsal markers chordin, otx2 and the ventral markers eve1, pax2.1, tall and gata1 between 75% epiboly and six-somites. From 24 hpf, morphants displayed an expansion of the ventral mesoderm-derived ICM, as evidenced by expansion of tall. Imo2 and crim1 itself. Analysis of the crim1 morphant phenotype in Tg(fli:EGFP) fish showed a clear reduction in the endothelial cells forming the intersegmental vessels and a loss of the dorsal longitudinal anastomotic vessel (DLAV). Hence, the primary role of zebrafish crim1 is likely to be the regulation of somitic and vascular development. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Scube2 mediates Hedgehog signalling in the zebrafish embryo

The Hedgehog family of secreted morphogens specifies the fate of a large number of different cell types within invertebrate and vertebrate embryos, including the muscle cell precursors of the embryonic myotome of zebrafish. Formation of Hedgehog-sensitive muscle fates is disrupted within homozygous zebrafish mutants of the you-type class, the majority of which disrupt components of the Hedgehog (HH) signal transduction pathway. We have undertaken a phenotypic and molecular characterisation of one of these mutants, you, which we show results from mutations within the zebrafish orthologue of the mammalian, gene scube2. This gene encodes a member of the Scube family of proteins, which is characterised by several protein motifs including EGF and CUB domains. Epistatic and molecular analyses position Scube2 function upstream of Smoothened (Smoh), the signalling component of the HH receptor complex, suggesting that Scube2 may act during HH signal transduction prior to, or during, receipt of the HH signal at the plasma membrane. In support of this model we show that scube2 has homology to cubilin, which encodes an endocytic receptor involved in protein trafficking suggesting a possible mode of function for Scube2 during HH signal transduction. (c) 2006 Elsevier Inc. All rights reserved.

Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo

Endothelial tip cells guide angiogenic sprouts by exploring the local environment for guidance cues such as vascular endothelial growth factor (VegfA). Here we present Flt1 (Vegf receptor 1) loss- and gain-of-function data in zebrafish showing that Flt1 regulates tip cell formation and arterial branching morphogenesis. Zebrafish embryos expressed soluble Flt1 (sFlt1) and membrane-bound Flt1 (mFlt1). In Tg(flt1(BAC):yfp) × Tg(kdrl:ras-cherry)(s916) embryos, flt1:yfp was expressed in tip, stalk and base cells of segmental artery sprouts and overlapped with kdrl:cherry expression in these domains. flt1 morphants showed increased tip cell numbers, enhanced angiogenic behavior and hyperbranching of segmental artery sprouts. The additional arterial branches developed into functional vessels carrying blood flow. In support of a functional role for the extracellular VEGF-binding domain of Flt1, overexpression of sflt1 or mflt1 rescued aberrant branching in flt1 morphants, and overexpression of sflt1 or mflt1 in controls resulted in short arterial sprouts with reduced numbers of filopodia. flt1 morphants showed reduced expression of Notch receptors and of the Notch downstream target efnb2a, and ectopic expression of flt4 in arteries, consistent with loss of Notch signaling. Conditional overexpression of the notch1a intracellular cleaved domain in flt1 morphants restored segmental artery patterning. The developing nervous system of the trunk contributed to the distribution of Flt1, and the loss of flt1 affected neurons. Thus, Flt1 acts in a Notch-dependent manner as a negative regulator of tip cell differentiation and branching. Flt1 distribution may be fine-tuned, involving interactions with the developing nervous system.

A study of the regulatory role of retinoic acid receptor gamma in Zebrafish development

Retinoic acid (RA) is thought to signal through retinoic acid receptors (RARs), i.e. RARα, β, and γ to play important roles in embryonic development and tissue regeneration. In this thesis, the zebrafish (Danio rario) was used as a vertebrate model organism to examine the role of RARγ. Treatment of zebrafish embryos with a RARγ specific agonist reduced the axial length of developing embryos, associated with reduced somite number and loss of hoxb13a expression. There were no clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist disrupted the formation of anterior structures of the head, the cranial bones and the anterior lateral line ganglia, associated with a loss of sox9 immunopositive cells in the same regions. Pectoral fin outgrowth was blocked by treatment with the RARγ agonist; however, this was not associated with loss of tbx5a immunopositive lateral plate cells and was reversed by wash out of the RARγ agonist or co-treatment with a RARγ antagonist. Regeneration of the transected caudal fin was also blocked by RARγ agonist treatment and restored by agonist washout or antagonist co-treatment; this phenotype was associated with a localised reduction in canonical Wnt signalling. Conversely, elevated canonical Wnt signalling after RARγ treatment was seen in other tissues, including ectopically in the notochord. Furthermore, some phenotypes seen in the RARγ treated embryos were present in mutant zebrafish embryos in which canonical Wnt signalling was constitutively increased. These data suggest that RARγ plays an essential role in maintaining neural crest and mesodermal stem/progenitor cells during normal embryonic development and tissue regeneration when the receptor is in its non-ligated state. In addition, this work has provided evidence that the activation status of RARγ may regulate hoxb13a gene expression and canonical Wnt signalling. Further research is required to confirm such novel regulatory roles.

Arsenic transport by zebrafish aquaglyceroporins

Background Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized. Results In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were namedaqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic. Conclusion This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

Contribution of lipophilic secondary metabolites to the toxicity of strains of freshwater cyanobacterial harmful algal blooms, identified using the zebrafish (Danio rerio) embryo as a model for vertebrate development

Cyanobacteria ("blue-green algae") are known to produce a diverse repertoire of biologically active secondary metabolites. When associated with so-called "harmful algal blooms", particularly in freshwater systems, a number of these metabolites have been associated—as "toxins", or commonly "cyanotoxins"—with human and animal health concerns. In addition to the known water-soluble toxins from these genera (i.e. microcystins, cylindrospermopsin, and saxitoxins), our studies have shown that there are metabolites within the lipophilic extracts of these strains that inhibit vertebrate development in zebrafish embryos. Following these studies, the zebrafish embryo model was implemented in the bioassay-guided purification of four isolates of cyanobacterial harmful algal blooms, namely Aphanizomenon, two isolates of Cylindrospermopsis, and Microcystis, in order to identify and chemically characterize the bioactive lipophilic metabolites in these isolates. ^ We have recently isolated a group of polymethoxy-1-alkenes (PMAs), as potential toxins, based on the bioactivity observed in the zebrafish embryos. Although PMAs have been previously isolated from diverse cyanobacteria, they have not previously been associated with relevant toxicity. These compounds seem to be widespread across the different genera of cyanobacteria, and, according to our studies, suggested to be derived from the polyketide biosynthetic pathway which is a common synthetic route for cyanobacterial and other algal toxins. Thus, it can be argued that these metabolites are perhaps important contributors to the toxicity of cyanobacterial blooms. In addition to the PMAs, a set of bioactive glycosidic carotenoids were also isolated because of their inhibition of zebrafish embryonic development. These pigmented organic molecules are found in many photosynthetic organisms, including cyanobacteria, and they have been largely associated with the prevention of photooxidative damage. This is the first indication of these compounds as toxic metabolites and the hypothesized mode of action is via their biotransformation to retinoids, some of which are known to be teratogenic. Additional fractions within all four isolates have been shown to contain other uncharacterized lipophilic toxic metabolites. This apparent repertoire of lipophilic compounds may contribute to the toxicity of these cyanobacterial harmful algal blooms, which were previously attributed primarily to the presence of the known water-soluble toxins.^

Quantitative assessment of intrinsic noise for visually guided behaviour in zebrafish

Supported by Royal Society of London (University Research Fellowship), Medical Research Council (New Investigator Research Grant) and CNRS.

CLARITY and PACT-based imaging of adult zebrafish and mouse for whole-animal analysis of infections.

Visualization of infection and the associated host response has been challenging in adult vertebrates. Owing to their transparency, zebrafish larvae have been used to directly observe infection in vivo; however, such larvae have not yet developed a functional adaptive immune system. Cells involved in adaptive immunity mature later and have therefore been difficult to access optically in intact animals. Thus, the study of many aspects of vertebrate infection requires dissection of adult organs or ex vivo isolation of immune cells. Recently, CLARITY and PACT (passive clarity technique) methodologies have enabled clearing and direct visualization of dissected organs. Here, we show that these techniques can be applied to image host-pathogen interactions directly in whole animals. CLARITY and PACT-based clearing of whole adult zebrafish and Mycobacterium tuberculosis-infected mouse lungs enables imaging of mycobacterial granulomas deep within tissue to a depth of more than 1 mm. Using established transgenic lines, we were able to image normal and pathogenic structures and their surrounding host context at high resolution. We identified the three-dimensional organization of granuloma-associated angiogenesis, an important feature of mycobacterial infection, and characterized the induction of the cytokine tumor necrosis factor (TNF) within the granuloma using an established fluorescent reporter line. We observed heterogeneity in TNF induction within granuloma macrophages, consistent with an evolving view of the tuberculous granuloma as a non-uniform, heterogeneous structure. Broad application of this technique will enable new understanding of host-pathogen interactions in situ.

The Role of Dopaminergic Systems in the Neurobehavioral Teratology of Organophosphates in Zebrafish

Background: Organophosphate (OP) pesticides are well-known developmental neurotoxicants that have been linked to abnormal cognitive and behavioral endpoints through both epidemiological studies and animal models of behavioral teratology, and are implicated in the dysfunction of multiple neurotransmitters, including dopamine. Chemical similarities between OP pesticides and organophosphate flame retardants (OPFRs), a class of compounds growing in use and environmental relevance, have produced concern regarding whether developmental exposures to OPFRs and OP pesticides may share behavioral outcomes, impacts on dopaminergic systems, or both. Methods: Using the zebrafish animal model, we exposed developing fish to two OPFRs, TDCIPP and TPHP, as well as the OP pesticide chlorpyrifos, during the first 5 days following fertilization. From there, the exposed fish were assayed for behavioral abnormalities and effects on monoamine neurochemistry as both larvae and adults. An experiment conducted in parallel examined how antagonism of the dopamine system during an identical window of development could alter later life behavior in the same assays. Finally, we investigated the interaction between developmental exposure to an OPFR and acute dopamine antagonism in larval behavior. Results: Developmental exposure to all three OP compounds altered zebrafish behavior, with effects persisting into adulthood. Additionally, exposure to an OPFR decreased the behavioral response to acute D2 receptor antagonism in larvae. However, the pattern of behavioral effects diverged substantially from those seen following developmental dopamine antagonism, and the investigations into dopamine neurochemistry were too variable to be conclusive. Thus, although the results support the hypothesis that OPFRs, as with OP pesticides such as chlorpyrifos, may present a risk to normal behavioral development, we were unable to directly link these effects to any dopaminergic dysfunction.

Citotoxicidade da associação de agrotóxicos da rizicultura em hepatócitos de Zebrafish

No plantio do arroz parte de um corpo d’água (rio, lago, lagoa) é desviado para a irrigação da plantação, e, posteriormente, a água utilizada nas lavouras é devolvida ao rio/lago/lagoa de origem. Assim, seja por lixiviação ou por qualquer outro fator, a água entra em contato com os agrotóxicos que, anteriormente, foram utilizados na plantação, podendo causar danos à qualidade do recurso hídrico e à fauna lacustre, devido à exposição a estes poluentes. O presente trabalho teve por objetivo verificar a citotoxicidade de agrotóxicos (herbicida e inseticida), utilizados na rizicultura no estado do Rio Grande do Sul, em células hepáticas da linhagem ZF-L. A partir da análise de funcionalidade de três alvos celulares diferentes, integridade da membrana celular, estabilidade lisossomal e atividade mitocondrial frente à exposição ao Roundup Transorb® , ao Furadan 350 SC® e à associação destes produtos. Foi analisada ainda, a capacidade de defesa das células, expostas aos poluentes escolhidos, no que diz respeito à atividade de proteínas extrusoras de xenobióticos, assim como à expressão de tais proteínas. A partir dos resultados obtidos foi verificado efeito citotóxico de ambos os agrotóxicos, bem como a mistura destes para todos os alvos verificados, apresentando ainda efeito inibitório à atividade de extrusão de xenobióticos pelas glicoproteínas P (P-gps). Apenas quando expostas ao inseticida e à mistura as células apresentaram um aumento na expressão de glicoproteínas (P-gp). Verificou-se a existência de correlação negativa entre a citotoxicidade apresentada, principalmente na atividade mitocondrial e na integridade lisossomo e a atividade das P-gps. Em conclusão, percebeu-se que as concentrações abaixo do permitido pela legislação brasileira, para os princípios ativos dos agrotóxicos testados, mostraram-se tóxicas para todos os alvos de citotoxicidade testados neste estudo, com exceção da mitocôndria, sugerindo que esta toxicidade apresentada pode ser devido aos surfactantes presentes nas formulações comerciais.

Telomeres in aging and disease: lessons from zebrafish

Age is the highest risk factor for some of the most prevalent human diseases, including cancer. Telomere shortening is thought to play a central role in the aging process in humans. The link between telomeres and aging is highlighted by the fact that genetic diseases causing telomerase deficiency are associated with premature aging and increased risk of cancer. For the last two decades, this link has been mostly investigated using mice that have long telomeres. However, zebrafish has recently emerged as a powerful and complementary model system to study telomere biology. Zebrafish possess human-like short telomeres that progressively decline with age, reaching lengths in old age that are observed when telomerase is mutated. The extensive characterization of its well-conserved molecular and cellular physiology makes this vertebrate an excellent model to unravel the underlying relationship between telomere shortening, tissue regeneration, aging and disease. In this Review, we explore the advantages of using zebrafish in telomere research and discuss the primary discoveries made in this model that have contributed to expanding our knowledge of how telomere attrition contributes to cellular senescence, organ dysfunction and disease.

X-Linked Retinitis Pigmentosa 2 Is a Novel Maternal-Effect Gene Required for Left-Right Asymmetry in Zebrafish

Retinitis pigmentosa 2 (RP2) gene is responsible for up to 20% of X-linked retinitis pigmentosa, a severe heterogeneous genetic disorder resulting in progressive retinal degeneration in humans. In vertebrates, several bodies of evidence have clearly established the role of Rp2 protein in cilia genesis and/or function. Unexpectedly, some observations in zebrafish have suggested the oocyte-predominant expression of the rp2 gene, a typical feature of maternal-effect genes. In the present study, we investigate the maternal inheritance of rp2 gene products in zebrafish eggs in order to address whether rp2 could be a novel maternal-effect gene required for normal development. Although both rp2 mRNA and corresponding protein are expressed during oogenesis, rp2 mRNA is maternally inherited, in contrast to Rp2 protein. A knockdown of the protein transcribed from both rp2 maternal and zygotic mRNA results in delayed epiboly and severe developmental defects, including eye malformations, that were not observed when only the protein from zygotic origin was knocked down. Moreover, the knockdown of maternal and zygotic Rp2 revealed a high incidence of left-right asymmetry establishment defects compared to only zygotic knockdown. Here we show that rp2 is a novel maternal-effect gene exclusively expressed in oocytes within the zebrafish ovary and demonstrate that maternal rp2 mRNA is essential for successful embryonic development and thus contributes to egg developmental competence. Our observations also reveal that Rp2 protein translated from maternal mRNA is important to allow normal heart loop formation, thus providing evidence of a direct maternal contribution to left-right asymmetry establishment.