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Sample records for normal zebrafish pronephros

  1. Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development

    PubMed Central

    Skouloudaki, Kassiani; Puetz, Michael; Simons, Matias; Courbard, Jean-Remy; Boehlke, Christopher; Hartleben, Björn; Engel, Christina; Moeller, Marcus J.; Englert, Christoph; Bollig, Frank; Schäfer, Tobias; Ramachandran, Haribaskar; Mlodzik, Marek; Huber, Tobias B.; Kuehn, E. Wolfgang; Kim, Emily; Kramer-Zucker, Albrecht; Walz, Gerd

    2009-01-01

    Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila. Vertebrates express 4 Fat molecules, Fat1–4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates. PMID:19439659

  2. Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta

    PubMed Central

    Gerlach, Gary F.; Wingert, Rebecca A.

    2014-01-01

    The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζ serve partially redundant functions to establish pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by

  3. Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta.

    PubMed

    Gerlach, Gary F; Wingert, Rebecca A

    2014-12-15

    The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζ serve partially redundant functions to establish pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by

  4. A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish

    PubMed Central

    Ichimura, Koichiro; Bubenshchikova, Ekaterina; Powell, Rebecca; Fukuyo, Yayoi; Nakamura, Tomomi; Tran, Uyen; Oda, Shoji; Tanaka, Minoru; Wessely, Oliver; Kurihara, Hidetake; Sakai, Tatsuo; Obara, Tomoko

    2012-01-01

    The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects. PMID:23028906

  5. The Na+/PO4 cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros in Xenopus and zebrafish embryos.

    PubMed

    Nichane, Massimo; Van Campenhout, Claude; Pendeville, Hélène; Voz, Marianne L; Bellefroid, Eric J

    2006-10-01

    The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na(+)/PO(4) cotransporter SLC20A1 (PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations.

  6. Loss of vhl in the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma.

    PubMed

    Noonan, Haley R; Metelo, Ana M; Kamei, Caramai N; Peterson, Randall T; Drummond, Iain A; Iliopoulos, Othon

    2016-08-01

    Patients with von Hippel-Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. 'Clear cell' tumors contain large, proliferating cells with 'clear cytoplasm', and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl(-/-)) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl(-/-) zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl(-/-) zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl(-/-) pronephros is reminiscent of clear cell histology, indicating that the vhl(-/-) mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl(-/-) zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC.

  7. Loss of vhl in the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma

    PubMed Central

    Noonan, Haley R.; Metelo, Ana M.; Kamei, Caramai N.; Peterson, Randall T.; Drummond, Iain A.

    2016-01-01

    ABSTRACT Patients with von Hippel–Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. ‘Clear cell’ tumors contain large, proliferating cells with ‘clear cytoplasm’, and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl−/−) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl−/− zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl−/− pronephros is reminiscent of clear cell histology, indicating that the vhl−/− mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl−/− zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC. PMID:27491085

  8. The tbx2a/b transcription factors direct pronephros segmentation and corpuscle of Stannius formation in zebrafish.

    PubMed

    Drummond, Bridgette E; Li, Yue; Marra, Amanda N; Cheng, Christina N; Wingert, Rebecca A

    2017-01-01

    The simplified and genetically conserved zebrafish pronephros is an excellent model to examine the cryptic processes of cell fate decisions during the development of nephron segments as well as the origins of associated endocrine cells that comprise the corpuscles of Stannius (CS). Using whole mount in situ hybridization, we found that transcripts of the zebrafish genes t-box 2a (tbx2a) and t-box 2b (tbx2b), which belong to the T-box family of transcription factors, were expressed in the caudal intermediate mesoderm progenitors that give rise to the distal pronephros and CS. Deficiency of tbx2a, tbx2b or both tbx2a/b reduced the size of the distal late (DL) segment, which was accompanied by a proximal convoluted segment (PCT) expansion. Further, tbx2a/b deficiency led to significantly larger CS clusters. These phenotypes were also observed in embryos with the from beyond (fby)(c144) mutation, which encodes a premature stop codon in the tbx2b T-box sequence. Conversely, overexpression of tbx2a and tbx2b in wild-type embryos expanded the DL segment where cells were comingled with the adjacent DE, and also decreased CS cell number, but notably did not alter PCT development-providing independent evidence that tbx2a and tbx2b are each necessary and sufficient to promote DL fate and suppress CS genesis. Epistasis studies indicated that tbx2a acts upstream of tbx2b to regulate the DL and CS fates, and likely has other targets as well. Retinoic acid (RA) addition and inhibition studies revealed that tbx2a and tbx2b are negatively regulated by RA signaling. Interestingly, the CS cell expansion that typifies tbx2a/b deficiency also occurred when blocking Notch signaling with the chemical DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). Ectopic activation of Notch in Tg(hsp70::Gal4; UAS::NICD)(NICD) embryos led to a reduced CS post heat-shock induction. To further examine the link between the tbx2a/b genes and Notch during CS formation, DAPT

  9. Evaluation of the teratogenic effects of three traditional Chinese medicines, Si Jun Zi Tang, Liu Jun Zi Tang and Shenling Baizhu San, during zebrafish pronephros development

    PubMed Central

    Ding, Yu-Ju; Wang, Bo-Cheng; Wen, Chi-Chung; Sun, Chiao-Yin; Lee, Hsun-Hua; Lee, Fei-Peng; Yang, Ling-Ling; Chen, Yau-Hung

    2015-01-01

    The aim of this study was to evaluate the teratogenic effects of three common Chinese medical prescriptions, Si Jun Zi Tang (SJZT), Liu Jun Zi Tang (LJZT) and Shenling Baizhu San (SLBS), during zebrafish pronephros development. We used the transgenic zebrafish line Tg(wt1b:EGFP) to assess the teratogenic effects using 12 different protocols, which comprised combinations of 4 doses (0, 25, 250, 1,250 ng/mL) and 3 exposure methods [methods I, 12–36 hours post fertilization (hpf), II, 24–48 hpf, and III, 24–36 hpf]. As a result, few defects in the kidneys were observed in the embryos exposed to 25 ng/mL of each medical prescription. The percentage of kidney malformation phenotypes increased as the exposure concentrations increased (25 ng/mL, 0–10%; 250 ng/mL, 0–60%; 1,250 ng/mL, 80–100%). Immunohistochemistry for α6F, which is a basolateral and renal tubular differentiation marker, revealed no obvious defective phenotypes in either SJZT- or LJZT-treated embryos, indicating that these Chinese medical prescriptions had minimal adverse effects on the pronephric duct. However, SLBS-treated embryos displayed a defective phenotype in the pronephric duct. According to these findings, we suggest (1) that the Chinese medical prescriptions induced kidney malformation phenotypes that are dose dependent and (2) that the embryonic zebrafish kidney was more sensitive to SLBS than SJZT and LJZT. PMID:26441476

  10. pdzrn3 is required for pronephros morphogenesis in Xenopus laevis.

    PubMed

    Marracci, Silvia; Vangelisti, Alberto; Raffa, Vittoria; Andreazzoli, Massimiliano; Dente, Luciana

    2016-01-01

    Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.

  11. Normal anatomy and histology of the adult zebrafish.

    PubMed

    Menke, Aswin L; Spitsbergen, Jan M; Wolterbeek, Andre P M; Woutersen, Ruud A

    2011-08-01

    The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

  12. Sept6 is required for ciliogenesis in Kupffer's vesicle, the pronephros, and the neural tube during early embryonic development.

    PubMed

    Zhai, Gang; Gu, Qilin; He, Jiangyan; Lou, Qiyong; Chen, Xiaowen; Jin, Xia; Bi, Erfei; Yin, Zhan

    2014-04-01

    Septins are conserved filament-forming GTP-binding proteins that act as cellular scaffolds or diffusion barriers in a number of cellular processes. However, the role of septins in vertebrate development remains relatively obscure. Here, we show that zebrafish septin 6 (sept6) is first expressed in the notochord and then in nearly all of the ciliary organs, including Kupffer's vesicle (KV), the pronephros, eye, olfactory bulb, and neural tube. Knockdown of sept6 in zebrafish embryos results in reduced numbers and length of cilia in KV. Consequently, cilium-related functions, such as the left-right patterning of internal organs and nodal/spaw signaling, are compromised. Knockdown of sept6 also results in aberrant cilium formation in the pronephros and neural tube, leading to cilium-related defects in pronephros development and Sonic hedgehog (Shh) signaling. We further demonstrate that SEPT6 associates with acetylated α-tubulin in vivo and localizes along the axoneme in the cilia of zebrafish pronephric duct cells as well as cultured ZF4 cells. Our study reveals a novel role of sept6 in ciliogenesis during early embryonic development in zebrafish.

  13. Sparc Protein Is Required for Normal Growth of Zebrafish Otoliths

    PubMed Central

    Kang, Young-Jin; Stevenson, Amy K.; Yau, Peter M.

    2008-01-01

    Otoliths and the homologous otoconia in the inner ear are essential for balance. Their morphogenesis is less understood than that of other biominerals, such as bone, and only a small number of their constituent proteins have been characterized. As a novel approach to identify unknown otolith proteins, we employed shotgun proteomics to analyze crude extracts from trout and catfish otoliths. We found three proteins that had not been associated previously with otolith or otoconia formation: ‘Secreted acidic cysteine rich glycoprotein’ (Sparc), an important bone protein that binds collagen and Ca2+; precerebellin-like protein, which contains a C1q domain and may associate with the collagenous otolin-1 during its assembly into a framework; and neuroserpin, a serine protease inhibitor that may regulate local protease activity during framework assembly. We then used the zebrafish to investigate whether Sparc plays a role in otolith morphogenesis. Immunodetection demonstrated that Sparc is a true constituent of otoliths. Knockdown of Sparc expression in morphant zebrafish resulted in four principal types of defective otoliths: smaller, extra and ectopic, missing and fused, or completely absent. Smaller size was the predominant phenotype and independent of the severity of otic-vesicle defects. These results suggested that Sparc is directly required for normal otolith growth. PMID:18784957

  14. Technical brief: Constant intense light exposure to lesion and initiate regeneration in normally pigmented zebrafish.

    PubMed

    Rajaram, Kamya; Summerbell, Emily R; Patton, James G

    2014-01-01

    Zebrafish are capable of robust and spontaneous regeneration of injured retina. Constant intense light exposure to adult albino zebrafish specifically causes apoptosis of rod and cone photoreceptor cells and is an excellent model to study the molecular mechanisms underlying photoreceptor regeneration. However, this paradigm has only been applied to lesion zebrafish of the nonpigmented albino genetic background, which precludes the use of numerous transgenic reporter lines that are widely used to study regeneration. Here, we explored the effectiveness of constant intense light exposure in causing photoreceptor apoptosis and stimulating regeneration in normally pigmented zebrafish retinas. We show that constant intense light exposure causes widespread photoreceptor damage in the dorsal-central retinas of pigmented zebrafish. Photoreceptor loss triggers dedifferentiation and proliferation of Müller glia as well as progenitor cell proliferation. We also demonstrate that the timeline of regeneration response is comparable between the albino and the pigmented retinas.

  15. Technical brief: Constant intense light exposure to lesion and initiate regeneration in normally pigmented zebrafish

    PubMed Central

    Rajaram, Kamya; Summerbell, Emily R.

    2014-01-01

    Zebrafish are capable of robust and spontaneous regeneration of injured retina. Constant intense light exposure to adult albino zebrafish specifically causes apoptosis of rod and cone photoreceptor cells and is an excellent model to study the molecular mechanisms underlying photoreceptor regeneration. However, this paradigm has only been applied to lesion zebrafish of the nonpigmented albino genetic background, which precludes the use of numerous transgenic reporter lines that are widely used to study regeneration. Here, we explored the effectiveness of constant intense light exposure in causing photoreceptor apoptosis and stimulating regeneration in normally pigmented zebrafish retinas. We show that constant intense light exposure causes widespread photoreceptor damage in the dorsal-central retinas of pigmented zebrafish. Photoreceptor loss triggers dedifferentiation and proliferation of Müller glia as well as progenitor cell proliferation. We also demonstrate that the timeline of regeneration response is comparable between the albino and the pigmented retinas. PMID:25324680

  16. Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

    PubMed Central

    Moore, John C.; Langenau, David M.

    2014-01-01

    Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish. PMID:25591079

  17. Inversin relays Frizzled-8 signals to promote proximal pronephros development

    PubMed Central

    Lienkamp, Soeren; Ganner, Athina; Boehlke, Christopher; Schmidt, Thorsten; Arnold, Sebastian J.; Schäfer, Tobias; Romaker, Daniel; Schuler, Julia; Hoff, Sylvia; Powelske, Christian; Eifler, Annekathrin; Krönig, Corinna; Bullerkotte, Axel; Nitschke, Roland; Kuehn, E. Wolfgang; Kim, Emily; Burkhardt, Hans; Brox, Thomas; Ronneberger, Olaf; Gloy, Joachim; Walz, Gerd

    2010-01-01

    Mutations of inversin cause type II nephronophthisis, an infantile autosomal recessive disease characterized by cystic kidney disease and developmental defects. Inversin regulates Wnt signaling and is required for convergent extension movements during early embryogenesis. We now show that Inversin is essential for Xenopus pronephros formation, involving two distinct and opposing forms of cell movements. Knockdown of Inversin abrogated both proximal pronephros extension and distal tubule differentiation, phenotypes similar to that of Xenopus deficient in Frizzled-8. Exogenous Inversin rescued the pronephric defects caused by lack of Frizzled-8, indicating that Inversin acts downstream of Frizzled-8 in pronephros morphogenesis. Depletion of Inversin prevents the recruitment of Dishevelled in response to Frizzled-8 and impeded the accumulation of Dishevelled at the apical membrane of tubular epithelial cells in vivo. Thus, defective tubule morphogenesis seems to contribute to the renal pathology observed in patients with nephronophthisis type II. PMID:21059920

  18. Standardized echocardiographic assessment of cardiac function in normal adult zebrafish and heart disease models.

    PubMed

    Wang, Louis W; Huttner, Inken G; Santiago, Celine F; Kesteven, Scott H; Yu, Ze-Yan; Feneley, Michael P; Fatkin, Diane

    2017-01-01

    The zebrafish (Danio rerio) is an increasingly popular model organism in cardiovascular research. Major insights into cardiac developmental processes have been gained by studies of embryonic zebrafish. However, the utility of zebrafish for modeling adult-onset heart disease has been limited by a lack of robust methods for in vivo evaluation of cardiac function. We established a physiological protocol for underwater zebrafish echocardiography using high frequency ultrasound, and evaluated its reliability in detecting altered cardiac function in two disease models. Serial assessment of cardiac function was performed in wild-type zebrafish aged 3 to 12 months and the effects of anesthetic agents, age, sex and background strain were evaluated. There was a varying extent of bradycardia and ventricular contractile impairment with different anesthetic drugs and doses, with tricaine 0.75 mmol l(-1) having a relatively more favorable profile. When compared with males, female fish were larger and had more measurement variability. Although age-related increments in ventricular chamber size were greater in females than males, there were no sex differences when data were normalized to body size. Systolic ventricular function was similar in both sexes at all time points, but differences in diastolic function were evident from 6 months onwards. Wild-type fish of both sexes showed a reliance on atrial contraction for ventricular diastolic filling. Echocardiographic evaluation of adult zebrafish with diphtheria toxin-induced myocarditis or anemia-induced volume overload accurately identified ventricular dilation and altered contraction, with suites of B-mode, ventricular strain, pulsed-wave Doppler and tissue Doppler indices showing concordant changes indicative of myocardial hypocontractility or hypercontractility, respectively. Repeatability, intra-observer and inter-observer correlations for echocardiographic measurements were high. We demonstrate that high frequency

  19. Standardized echocardiographic assessment of cardiac function in normal adult zebrafish and heart disease models

    PubMed Central

    Wang, Louis W.; Huttner, Inken G.; Santiago, Celine F.; Kesteven, Scott H.; Yu, Ze-Yan; Feneley, Michael P.

    2017-01-01

    ABSTRACT The zebrafish (Danio rerio) is an increasingly popular model organism in cardiovascular research. Major insights into cardiac developmental processes have been gained by studies of embryonic zebrafish. However, the utility of zebrafish for modeling adult-onset heart disease has been limited by a lack of robust methods for in vivo evaluation of cardiac function. We established a physiological protocol for underwater zebrafish echocardiography using high frequency ultrasound, and evaluated its reliability in detecting altered cardiac function in two disease models. Serial assessment of cardiac function was performed in wild-type zebrafish aged 3 to 12 months and the effects of anesthetic agents, age, sex and background strain were evaluated. There was a varying extent of bradycardia and ventricular contractile impairment with different anesthetic drugs and doses, with tricaine 0.75 mmol l−1 having a relatively more favorable profile. When compared with males, female fish were larger and had more measurement variability. Although age-related increments in ventricular chamber size were greater in females than males, there were no sex differences when data were normalized to body size. Systolic ventricular function was similar in both sexes at all time points, but differences in diastolic function were evident from 6 months onwards. Wild-type fish of both sexes showed a reliance on atrial contraction for ventricular diastolic filling. Echocardiographic evaluation of adult zebrafish with diphtheria toxin-induced myocarditis or anemia-induced volume overload accurately identified ventricular dilation and altered contraction, with suites of B-mode, ventricular strain, pulsed-wave Doppler and tissue Doppler indices showing concordant changes indicative of myocardial hypocontractility or hypercontractility, respectively. Repeatability, intra-observer and inter-observer correlations for echocardiographic measurements were high. We demonstrate that high

  20. Development of the zebrafish mesonephros

    PubMed Central

    Diep, Cuong Q.; Peng, Zhenzhen; Ukah, Tobechukwu K.; Kelly, Paul M.; Daigle, Renee V.; Davidson, Alan J.

    2015-01-01

    The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. PMID:25677367

  1. Cdc42 and sec10 Are Required for Normal Retinal Development in Zebrafish.

    PubMed

    Choi, Soo Young; Baek, Jeong-In; Zuo, Xiaofeng; Kim, Seok-Hyung; Dunaief, Joshua L; Lipschutz, Joshua H

    2015-05-01

    To characterize the function and mechanisms of cdc42 and sec10 in eye development in zebrafish. Knockdown of zebrafish cdc42 and sec10 was carried out using antisense morpholino injection. The phenotype of morphants was characterized by histology, immunohistology, and transmission electron microscopy (TEM). To investigate a synergistic genetic interaction between cdc42 and sec10, we titrated suboptimal doses of cdc42 and sec10 morpholinos, and coinjected both morpholinos. To study trafficking, a melanosome transport assay was performed using epinephrine. Cdc42 and sec10 knockdown in zebrafish resulted in both abnormal eye development and increased retinal cell death. Cdc42 morphants had a relatively normal retinal structure, aside from the absence of most connecting cilia and outer segments, whereas in sec10 morphants, much of the outer nuclear layer, which is composed of the photoreceptor nuclei, was missing and RPE cell thickness was markedly irregular. Knockdown of cdc42 and sec10 also resulted in an intracellular transport defect affecting retrograde melanosome transport. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 act in the same pathway in retinal development. We propose a model whereby sec10 and cdc42 play a central role in development of the outer segment of the retinal photoreceptor cell by trafficking proteins necessary for ciliogenesis.

  2. Cdc42 and sec10 Are Required for Normal Retinal Development in Zebrafish

    PubMed Central

    Choi, Soo Young; Baek, Jeong-In; Zuo, Xiaofeng; Kim, Seok-Hyung; Dunaief, Joshua L.; Lipschutz, Joshua H.

    2015-01-01

    Purpose. To characterize the function and mechanisms of cdc42 and sec10 in eye development in zebrafish. Methods. Knockdown of zebrafish cdc42 and sec10 was carried out using antisense morpholino injection. The phenotype of morphants was characterized by histology, immunohistology, and transmission electron microscopy (TEM). To investigate a synergistic genetic interaction between cdc42 and sec10, we titrated suboptimal doses of cdc42 and sec10 morpholinos, and coinjected both morpholinos. To study trafficking, a melanosome transport assay was performed using epinephrine. Results. Cdc42 and sec10 knockdown in zebrafish resulted in both abnormal eye development and increased retinal cell death. Cdc42 morphants had a relatively normal retinal structure, aside from the absence of most connecting cilia and outer segments, whereas in sec10 morphants, much of the outer nuclear layer, which is composed of the photoreceptor nuclei, was missing and RPE cell thickness was markedly irregular. Knockdown of cdc42 and sec10 also resulted in an intracellular transport defect affecting retrograde melanosome transport. Furthermore, there was a synergistic genetic interaction between zebrafish cdc42 and sec10, suggesting that cdc42 and sec10 act in the same pathway in retinal development. Conclusions. We propose a model whereby sec10 and cdc42 play a central role in development of the outer segment of the retinal photoreceptor cell by trafficking proteins necessary for ciliogenesis. PMID:26024121

  3. Evi1 is specifically expressed in the distal tubule and duct of the Xenopus pronephros and plays a role in its formation.

    PubMed

    Van Campenhout, Claude; Nichane, Massimo; Antoniou, Aline; Pendeville, Hélène; Bronchain, Odile J; Marine, Jean-Christophe; Mazabraud, Andre; Voz, Marianne L; Bellefroid, Eric J

    2006-06-01

    The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus, Evi1 and MEL1 have partially overlapping restricted embryonic expression profiles. Within the pronephros, Evi1 and MEL1 are sequentially expressed within the distal tubule and duct compartments, Evi1 transcription being detected prior to any sign of pronephric morphogenesis. In the pronephros of zebrafish embryos, Evi1 expression is restricted to the posterior portion of the duct, the anterior portion having characteristics of proximal tubules. In the Xenopus pronephros, Evi1 expression is upregulated by retinoid signaling and repressed by overexpression of xWT1 and by Notch signaling. Overexpression of Evi1 from late neurula stage specifically inhibits the expression of proximal tubule and glomus pronephric markers. We show that the first zinc finger and CtBP interaction domains are required for this activity. Overexpression of a hormone-inducible Evi1-VP16 antimorphic fusion with activation at neurula stage disrupts distal tubule and duct formation and expands the expression of glomus markers. Although overexpression of this construct also causes in many embryos a reduction of proximal tubule markers, embryos with expanded and ectopic staining have been also observed. Together, these data indicate that Evi1 plays a role in the proximo-distal patterning of the pronephros and suggest that it may do so by functioning as a CtBP dependent repressor.

  4. High-precision registration between zebrafish brain atlases using symmetric diffeomorphic normalization.

    PubMed

    Marquart, Gregory D; Tabor, Kathryn M; Horstick, Eric J; Brown, Mary; Geoca, Alexandra K; Polys, Nicholas F; Nogare, Damian Dalle; Burgess, Harold A

    2017-08-01

    Atlases provide a framework for spatially mapping information from diverse sources into a common reference space. Specifically, brain atlases allow annotation of gene expression, cell morphology, connectivity, and activity. In larval zebrafish, advances in genetics, imaging, and computational methods now allow the collection of such information brain-wide. However, due to technical considerations, disparate datasets may use different references and may not be aligned to the same coordinate space. Two recent larval zebrafish atlases exemplify this problem: Z-Brain, containing gene expression, neural activity, and neuroanatomical segmentations, was acquired using immunohistochemical stains, while the Zebrafish Brain Browser (ZBB) was constructed from live scans of fluorescent reporters in transgenic larvae. Although different references were used, the atlases included several common transgenic patterns that provide potential "bridges" for transforming each into the other's coordinate space. We tested multiple bridging channels and registration algorithms and found that the symmetric diffeomorphic normalization algorithm improved live brain registration precision while better preserving cell morphology than B-spline-based registrations. Symmetric diffeomorphic normalization also corrected for tissue distortion introduced during fixation. Multi-reference channel optimization provided a transformation that enabled Z-Brain and ZBB to be co-aligned with precision of approximately a single cell diameter and minimal perturbation of cell and tissue morphology. Finally, we developed software to visualize brain regions in 3 dimensions, including a virtual reality neuroanatomy explorer. This study demonstrates the feasibility of integrating whole brain datasets, despite disparate reference templates and acquisition protocols, when sufficient information is present for bridging. Increased accuracy and interoperability of zebrafish digital brain atlases will facilitate

  5. Expression of Wnt Signaling Components during Xenopus Pronephros Development

    PubMed Central

    Zhang, Bo; Tran, Uyen; Wessely, Oliver

    2011-01-01

    Background The formation of the vertebrate kidney is tightly regulated and relies on multiple evolutionarily conserved inductive events. These are present in the complex metanephric kidney of higher vertebrates, but also in the more primitive pronephric kidney functional in the larval stages of amphibians and fish. Wnts have long been viewed as central in this process. Canonical β-Catenin-dependent Wnt signaling establishes kidney progenitors and non-canonical β-Catenin-independent Wnt signaling participate in the morphogenetic processes that form the highly sophisticated nephron structure. While some individual Wnt signaling components have been studied extensively in the kidney, the overall pathway has not yet been analyzed in depth. Methodology/Principal Findings Here we report a detailed expression analysis of all Wnt ligands, receptors and several downstream Wnt effectors during pronephros development in Xenopus laevis using in situ hybridization. Out of 19 Wnt ligands, only three, Wnt4, Wnt9a and Wnt11, are specifically expressed in the pronephros. Others such as Wnt8a are present, but in a broader domain comprising adjacent tissues in addition to the kidney. The same paradigm is observed for the Wnt receptors and its downstream signaling components. Fzd1, Fzd4, Fzd6, Fzd7, Fzd8 as well as Celsr1 and Prickle1 show distinct expression domains in the pronephric kidney, whereas the non-traditional Wnt receptors, Ror2 and Ryk, as well as the majority of the effector molecules are rather ubiquitous. In addition to this spatial regulation, the timing of expression is also tightly regulated. In particular, non-canonical Wnt signaling seems to be restricted to later stages of pronephros development. Conclusion/Significance Together these data suggest a complex cross talk between canonical and non-canonical Wnt signaling is required to establish a functional pronephric kidney. PMID:22028899

  6. The Ciliopathy Gene ahi1 Is Required for Zebrafish Cone Photoreceptor Outer Segment Morphogenesis and Survival

    PubMed Central

    Lessieur, Emma M.; Fogerty, Joseph; Gaivin, Robert J.; Song, Ping; Perkins, Brian D.

    2017-01-01

    Purpose Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes. Methods Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs). Expression analysis was performed by whole-mount in situ hybridization. Anatomic and molecular characterization of photoreceptors was investigated by histology, electron microscopy, and immunohistochemistry. The optokinetic response (OKR) behavior assay was used to assess visual function. Kidney cilia were evaluated by whole-mount immunostaining. Results The ahi1lri46 mutation in zebrafish resulted in shorter cone outer segments but did not affect visual behavior at 5 days after fertilization (dpf). No defects in rod morphology or rhodopsin localization were observed at 5 dpf. By 5 months of age, cone degeneration and rhodopsin mislocalization in rod photoreceptors was observed. The connecting cilium formed normally and Cc2d2a and Cep290 localized properly. Distal pronephric duct cilia were absent in mutant fish; however, only 9% of ahi1 mutants had kidney cysts by 5 dpf, suggesting that the pronephros remained largely functional. Conclusions The results indicate that Ahi1 is required for photoreceptor disc morphogenesis and outer segment maintenance in zebrafish. PMID:28118669

  7. Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.

    PubMed

    Moore, John C; Tang, Qin; Yordán, Nora Torres; Moore, Finola E; Garcia, Elaine G; Lobbardi, Riadh; Ramakrishnan, Ashwin; Marvin, Dieuwke L; Anselmo, Anthony; Sadreyev, Ruslan I; Langenau, David M

    2016-11-14

    Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution. © 2016 Moore et al.

  8. Chordin Affects Pronephros Development in Xenopus Embryos by Anteriorizing Presomitic Mesoderm

    PubMed Central

    Mitchell, Tracy; Jones, Elizabeth A.; Weeks, Daniel L.; Sheets, Michael D.

    2007-01-01

    Spemann’s organizer emits signals that pattern the mesodermal germ layer during Xenopus embryogenesis. In a previous study, we demonstrated that FGFR1 activity within the organizer is required for the production of both the somitic muscle- and pronephros-patterning signals by the organizer and the expression of chordin, an organizer-specific secreted protein (Mitchell and Sheets [2001] Dev. Biol. 237:295-305). Studies from others in both chicken and Xenopus embryos provide compelling evidence that pronephros forms by means of secondary induction signals emitted from anterior somites (Seufert et al. [1999] Dev. Biol. 215:233-242; Mauch et al. [2000] Dev. Biol. 220:62-75). Here we provide several lines of evidence in support of the hypothesis that chordin influences pronephros development by directing the formation of anterior somites. Chordin mRNA was absent in ultraviolet (UV)-irradiated embryos lacking pronepheros (average DAI<2) but was always found in UV-irradiated embryos that retain pronepheros (average DAI>2). Furthermore, ectopic expression of chordin in embryos and in tissue explants leads to the formation of anterior somites and pronephros. In these experiments, pronephros was only observed in association with muscle. Chordin diverted somatic muscle cells to more anterior positions within the somite file in chordin-induced secondary trunks and induced the expression of the anterior myogenic gene myf5. Finally, depletion of chordin mRNA with DEED antisense oligonucleotides substantially reduced somitic muscle and pronephric tubule and duct formation in whole embryos. These data and previous studies on ectoderm and endoderm (Sasai et al. [1995] Nature 377:757) support the idea that chordin functions as an anteriorizing signal in patterning the germ layers during vertebrate embryogenesis. Our data support the hypothesis that chordin directs the formation of anterior somites that in turn are necessary for pronephros development. PMID:17106888

  9. TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish

    PubMed Central

    Opitz, Robert; Maquet, Emilie; Zoenen, Maxime; Dadhich, Rajesh

    2011-01-01

    TSH is the primary physiological regulator of thyroid gland function. The effects of TSH on thyroid cells are mediated via activation of its membrane receptor [TSH receptor (TSHR)]. In this study, we examined functional thyroid differentiation in zebrafish and characterized the role of TSHR signaling during thyroid organogenesis. Cloning of a cDNA encoding zebrafish Tshr showed conservation of primary structure and functional properties between zebrafish and mammalian TSHR. In situ hybridization confirmed that the thyroid is the major site of tshr expression during zebrafish development. In addition, we identified tpo, iyd, duox, and duoxa as novel thyroid differentiation markers in zebrafish. Temporal analyses of differentiation marker expression demonstrated the induction of an early thyroid differentiation program along with thyroid budding, followed by a delayed onset of duox and duoxa expression coincident with thyroid hormone synthesis. Furthermore, comparative analyses in mouse and zebrafish revealed for the first time a thyroid-enriched expression of cell death regulators of the B-cell lymphoma 2 family during early thyroid morphogenesis. Knockdown of tshr function by morpholino microinjection into embryos did not affect early thyroid morphogenesis but caused defects in later functional differentiation. The thyroid phenotype observed in tshr morphants at later stages comprised a reduction in number and size of functional follicles, down-regulation of differentiation markers, as well as reduced thyroid transcription factor expression. A comparison of our results with phenotypes observed in mouse models of defective TSHR and cAMP signaling highlights the value of zebrafish as a model to enhance the understanding of functional differentiation in the vertebrate thyroid. PMID:21737742

  10. Pescadillo homologue 1 and Peter Pan function during Xenopus laevis pronephros development.

    PubMed

    Tecza, Aleksandra; Bugner, Verena; Kühl, Michael; Kühl, Susanne J

    2011-10-01

    pes1 (pescadillo homologue 1) and ppan (Peter Pan) are multifunctional proteins involved in ribosome biogenesis, cell proliferation, apoptosis, cell migration and regulation of gene expression. Both proteins are required for early neural development in Xenopus laevis, as previously demonstrated. We show that the expression of both genes in the developing pronephros depends on wnt4 and fzd3 (frizzled homologue 3) function. Loss of pes1 or ppan by MO (morpholino oligonucleotide)-based knockdown approaches resulted in strong malformations during pronephric tubule formation. Defects were already notable during specification of pronephric progenitor cells, as shown by lhx1 expression. Moreover, we demonstrated that Xenopus pes1 and ppan interact physically and functionally and that pes1 and ppan can cross-rescue the loss of function phenotype of one another. Interference with rRNA synthesis, however, did not result in a similar early pronephros phenotype. These results demonstrate that pes1 and ppan are required for Xenopus pronephros development and indicate that their function in the pronephros is independent of their role in ribosome biosynthesis.

  11. Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish

    PubMed Central

    Garcia, Elaine G.; Lobbardi, Riadh; Jain, Esha; Tang, Qin; Moore, John C.; Cortes, Mauricio; Molodtsov, Aleksey; Kasheta, Melissa; Luo, Christina C.; Garcia, Amaris J.; Mylvaganam, Ravi; Yoder, Jeffrey A.; Blackburn, Jessica S.; Sadreyev, Ruslan I.; Ceol, Craig J.; North, Trista E.

    2016-01-01

    Hematopoiesis culminates in the production of functionally heterogeneous blood cell types. In zebrafish, the lack of cell surface antibodies has compelled researchers to use fluorescent transgenic reporter lines to label specific blood cell fractions. However, these approaches are limited by the availability of transgenic lines and fluorescent protein combinations that can be distinguished. Here, we have transcriptionally profiled single hematopoietic cells from zebrafish to define erythroid, myeloid, B, and T cell lineages. We also used our approach to identify hematopoietic stem and progenitor cells and a novel NK-lysin 4+ cell type, representing a putative cytotoxic T/NK cell. Our platform also quantified hematopoietic defects in rag2E450fs mutant fish and showed that these fish have reduced T cells with a subsequent expansion of NK-lysin 4+ cells and myeloid cells. These data suggest compensatory regulation of the innate immune system in rag2E450fs mutant zebrafish. Finally, analysis of Myc-induced T cell acute lymphoblastic leukemia showed that cells are arrested at the CD4+/CD8+ cortical thymocyte stage and that a subset of leukemia cells inappropriately reexpress stem cell genes, including bmi1 and cmyb. In total, our experiments provide new tools and biological insights into single-cell heterogeneity found in zebrafish blood and leukemia. PMID:27139488

  12. Wnt5a Is Necessary for Normal Kidney Development in Zebrafish and Mice

    PubMed Central

    Huang, Liwei; Xiao, An; Choi, Soo Young; Kan, Quane; Zhou, Weibin; Chacon-Heszele, Maria F.; Ryu, Yun Kyoung; McKenna, Sarah; Zuo, Xiaofeng; Kuruvilla, Rejji; Lipschutz, Joshua H.

    2015-01-01

    Background Wnt5a is important for the development of various organs and postnatal cellular function. Little is known, however, about the role of Wnt5a in kidney development, although WNT5A mutations were identified in patients with Robinow syndrome, a genetic disease which includes developmental defects in kidneys. Our goal in this study was to determine the role of Wnt5a in kidney development. Methods Whole-mount in situ hybridization was used to establish the expression pattern of Wnt5a during kidney development. Zebrafish with wnt5a knockdown and Wnt5a global knockout mice were used to identify kidney phenotypes. Results In zebrafish, wnt5a knockdown resulted in glomerular cyst formation and dilated renal tubules. In mice, Wnt5a global knockout resulted in pleiotropic, but severe, kidney phenotypes, including agenesis, fused kidney, hydronephrosis and duplex kidney/ureter. Conclusions Our data demonstrated the important role of Wnt5a in kidney development. Disrupted Wnt5a resulted in kidney cysts in zebrafish and pleiotropic abnormal kidney development in mice. PMID:25412793

  13. Wnt5a is necessary for normal kidney development in zebrafish and mice.

    PubMed

    Huang, Liwei; Xiao, An; Choi, Soo Young; Kan, Quane; Zhou, Weibin; Chacon-Heszele, Maria F; Ryu, Yun Kyoung; McKenna, Sarah; Zuo, Xiaofeng; Kuruvilla, Rejji; Lipschutz, Joshua H

    2014-01-01

    Wnt5a is important for the development of various organs and postnatal cellular function. Little is known, however, about the role of Wnt5a in kidney development, although WNT5A mutations were identified in patients with Robinow syndrome, a genetic disease which includes developmental defects in kidneys. Our goal in this study was to determine the role of Wnt5a in kidney development. Whole-mount in situ hybridization was used to establish the expression pattern of Wnt5a during kidney development. Zebrafish with wnt5a knockdown and Wnt5a global knockout mice were used to identify kidney phenotypes. In zebrafish, wnt5a knockdown resulted in glomerular cyst formation and dilated renal tubules. In mice, Wnt5a global knockout resulted in pleiotropic, but severe, kidney phenotypes, including agenesis, fused kidney, hydronephrosis and duplex kidney/ureter. Our data demonstrated the important role of Wnt5a in kidney development. Disrupted Wnt5a resulted in kidney cysts in zebrafish and pleiotropic abnormal kidney development in mice. © 2014 S. Karger AG, Basel.

  14. Zebrafish TARP Cacng2 is required for the expression and normal development of AMPA receptors at excitatory synapses.

    PubMed

    Roy, Birbickram; Ahmed, Kazi T; Cunningham, Marcus E; Ferdous, Jannatul; Mukherjee, Rajarshi; Zheng, Wang; Chen, Xing-Zhen; Ali, Declan W

    2016-05-01

    Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPA receptor-related proteins, known as TARPs. Little is known about the role of TARPs during development, or about their function in non-mammalian organisms. Here we report the presence of TARPs, specifically the prototypical TARP, stargazin, in developing zebrafish. We find that zebrafish express two forms of stargazin, Cacng2a and Cacng2b from as early as 12-h post fertilization (hpf). Knockdown of Cacng2a and Cacng2b via splice-blocking morpholinos resulted in embryos that exhibited deficits in C-start escape responses, showing reduced C-bend angles, smaller tail velocities and aberrant C-bend turning directions. Injection of the morphants with Cacng2a or 2b mRNA rescued the morphological phenotype and the synaptic deficits. To investigate the effect of reduced Cacng2a and 2b levels on synaptic physiology, we performed whole cell patch clamp recordings of AMPA mEPSCs from zebrafish Mauthner cells. Knockdown of Cacng2a results in reduced AMPA currents and lower mEPSC frequencies, whereas knockdown of Cacng2b displayed no significant change in mEPSC amplitude or frequency. Non-stationary fluctuation analysis confirmed a reduction in the number of active synaptic receptors in the Cacng2a but not in the Cacng2b morphants. Together, these results suggest that Cacng2a is required for normal trafficking and function of synaptic AMPARs, while Cacng2b is largely non-functional with respect to the development of AMPA synaptic transmission.

  15. Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development

    PubMed Central

    Marra, Amanda N.; Wingert, Rebecca A.

    2016-01-01

    Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a “salt-and-pepper” fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA

  16. Notch activates Wnt-4 signalling to control medio-lateral patterning of the pronephros.

    PubMed

    Naylor, Richard W; Jones, Elizabeth A

    2009-11-01

    Previous studies have highlighted a role for the Notch signalling pathway during pronephrogenesis in the amphibian Xenopus laevis, and in nephron development in the mammalian metanephros, yet a mechanism for this function remains elusive. Here, we further the understanding of how Notch signalling patterns the early X. laevis pronephros anlagen, a function that might be conserved in mammalian nephron segmentation. Our results indicate that early phase pronephric Notch signalling patterns the medio-lateral axis of the dorso-anterior pronephros anlagen, permitting the glomus and tubules to develop in isolation. We show that this novel function acts through the Notch effector gene hrt1 by upregulating expression of wnt4. Wnt-4 then patterns the proximal pronephric anlagen to establish the specific compartments that span the medio-lateral axis. We also identified pronephric expression of lunatic fringe and radical fringe that is temporally and spatially appropriate for a role in regulating Notch signalling in the dorso-anterior region of the pronephros anlagen. On the basis of these results, along with data from previous publications, we propose a mechanism by which the Notch signalling pathway regulates a Wnt-4 function that patterns the proximal pronephric anlagen.

  17. N-Ethylmaleimide–Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium

    PubMed Central

    Hanovice, Nicholas J.; Daly, Christina M. S.; Gross, Jeffrey M.

    2015-01-01

    Purpose Despite the number of albinism-causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has been identified, suggesting that our understanding of melanogenesis is incomplete. Previously, we identified two oculocutaneous albinism mutations in zebrafish, au13 and au18. Here, we sought to identify the mutated loci and determine how the affected proteins contribute to normal pigmentation of the retinal pigment epithelium (RPE). Methods Complementation analyses revealed that au13 and au18 belonged to a single complementation group, suggesting that they affected the same locus. Whole-genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to identify putative mutations, which were confirmed by cDNA sequencing and mRNA rescue. Transmission electron microscopy (TEM) and image quantification were used to identify the cellular basis of hypopigmentation. Results Whole-genome sequencing and SNP mapping identified a nonsense mutation in the N-ethylmaleimide–sensitive factor b (nsfb) gene in au18 mutants. Complementary DNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X). No coding sequence mutations were identified in au13, but quantitative PCR revealed a significant decrease in nsfb expression, and nsfb mRNA injection rescued the hypopigmentation phenotype, suggesting a regulatory mutation. In situ hybridization revealed that nsfb is broadly expressed during embryonic development, including in the RPE. Transmission electron microscopy analyses indicated that average melanosome density and maturity were significantly decreased in nsfb mutants. Conclusions au18 and au13 contain mutations in nsfb, which encodes a protein that is required for the maturation of melanosomes in zebrafish RPE. PMID:26618645

  18. N-Ethylmaleimide-Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium.

    PubMed

    Hanovice, Nicholas J; Daly, Christina M S; Gross, Jeffrey M

    2015-11-01

    Despite the number of albinism-causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has been identified, suggesting that our understanding of melanogenesis is incomplete. Previously, we identified two oculocutaneous albinism mutations in zebrafish, au13 and au18. Here, we sought to identify the mutated loci and determine how the affected proteins contribute to normal pigmentation of the retinal pigment epithelium (RPE). Complementation analyses revealed that au13 and au18 belonged to a single complementation group, suggesting that they affected the same locus. Whole-genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to identify putative mutations, which were confirmed by cDNA sequencing and mRNA rescue. Transmission electron microscopy (TEM) and image quantification were used to identify the cellular basis of hypopigmentation. Whole-genome sequencing and SNP mapping identified a nonsense mutation in the N-ethylmaleimide-sensitive factor b (nsfb) gene in au18 mutants. Complementary DNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X). No coding sequence mutations were identified in au13, but quantitative PCR revealed a significant decrease in nsfb expression, and nsfb mRNA injection rescued the hypopigmentation phenotype, suggesting a regulatory mutation. In situ hybridization revealed that nsfb is broadly expressed during embryonic development, including in the RPE. Transmission electron microscopy analyses indicated that average melanosome density and maturity were significantly decreased in nsfb mutants. au18 and au13 contain mutations in nsfb, which encodes a protein that is required for the maturation of melanosomes in zebrafish RPE.

  19. Heat Shock 70-kDa Protein 5 (Hspa5) Is Essential for Pronephros Formation by Mediating Retinoic Acid Signaling*

    PubMed Central

    Shi, Weili; Xu, Gang; Wang, Chengdong; Sperber, Steven M.; Chen, Yonglong; Zhou, Qin; Deng, Yi; Zhao, Hui

    2015-01-01

    Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family. As a multifunctional protein, it participates in protein folding and calcium homeostasis and serves as an essential regulator of the endoplasmic reticulum (ER) stress response. It has also been implicated in signal transduction by acting as a receptor or co-receptor residing at the plasma membrane. Its function during embryonic development, however, remains largely elusive. In this study, we used morpholino antisense oligonucleotides (MOs) to knock down Hspa5 activity in Xenopus embryos. In Hspa5 morphants, pronephros formation was strongly inhibited with the reduction of pronephric marker genes Lim homeobox protein 1 (lhx1), pax2, and β1 subunit of Na/K-ATPase (atp1b1). Pronephros tissue was induced in vitro by treating animal caps with all-trans-retinoic acid and activin. Depletion of Hspa5 in animal caps, however, blocked the induction of pronephros as well as reduced the expression of retinoic acid (RA)-responsive genes, suggesting that knockdown of Hspa5 attenuated RA signaling. Knockdown of Hspa5 in animal caps resulted in decreased expression of lhx1, a transcription factor directly regulated by RA signaling and essential for pronephros specification. Co-injection of Hspa5MO with lhx1 mRNA partially rescued the phenotype induced by Hspa5MO. These results suggest that the RA-Lhx1 signaling cascade is involved in Hspa5MO-induced pronephros malformation. This study shows that Hspa5, a key regulator of the unfolded protein response, plays an essential role in pronephros formation, which is mediated in part through RA signaling during early embryonic development. PMID:25398881

  20. Short-term overfeeding of zebrafish with normal or high-fat diet as a model for the development of metabolically healthy versus unhealthy obesity.

    PubMed

    Landgraf, Kathrin; Schuster, Susanne; Meusel, Andrej; Garten, Antje; Riemer, Thomas; Schleinitz, Dorit; Kiess, Wieland; Körner, Antje

    2017-03-21

    Obese individuals differ in their risk of developing metabolic and cardiovascular complications depending on fat distribution (subcutaneous versus visceral) and adipose tissue (AT) phenotype (hyperplasic versus hypertrophic). However, the exact mechanisms which determine whether an obese individual is metabolically healthy or unhealthy are not clear, and analyses of the underlying pathomechanisms are limited by the lack of suitable in vivo models in which metabolically healthy versus metabolically unhealthy AT accumulation can be specifically induced. In the current study, we aimed to establish a protocol for the use of zebrafish as a model for obesity-related metabolically healthy versus metabolically unhealthy AT accumulation. We overfed adult male zebrafish of the AB strain with normal fat diet (NFD) or high fat diet (HFD) for 8 weeks and compared parameters related to obesity, i.e. body weight, body mass index, condition index and body fat percentage, to control zebrafish fed under physiological conditions. In addition, we investigated the presence of early obesity-related metabolic alterations by quantifying blood glucose levels, plasma triglyceride and cholesterol levels, and by assessing ectopic lipid accumulation in the liver of zebrafish. Finally, we determined gene expression levels of marker genes related to lipid metabolism, inflammation and fibrosis in visceral AT and liver. We show that 8-weeks overfeeding with either NFD or HFD leads to a significant increase in body weight and AT mass compared to controls. In contrast to NFD-overfed zebrafish, HFD-overfed zebrafish additionally present metabolic alterations, e.g. hyperglycemia and ectopic lipid accumulation in the liver, and a metabolically unhealthy AT phenotype with adipocyte hypertrophy especially in the visceral AT depot, which is accompanied by changes in the expression of marker genes for lipid metabolism, inflammation and fibrosis. In summary, we have established a method for the specific

  1. Multiple ribosomal proteins are expressed at high levels in developing zebrafish endoderm and are required for normal exocrine pancreas development.

    PubMed

    Provost, Elayne; Weier, Christopher A; Leach, Steven D

    2013-06-01

    Ribosomal protein L (rpl) genes are essential for assembly of the 60S subunit of the eukaryotic ribosome and may also carry out additional extra-ribosomal functions. We have identified a common expression pattern for rpl genes in developing zebrafish larvae. After initially widespread expression in early embryos, the expression of multiple rpl genes becomes increasingly restricted to the endoderm. With respect to the pancreas, rpl genes are highly expressed in ptf1a-expressing pancreatic progenitors at 48 hpf, suggesting possible functional roles in pancreatic morphogenesis and/or differentiation. Utilizing two available mutant lines, rpl23a(hi2582) and rpl6(hi3655b), we found that ptf1a-expressing pancreatic progenitors fail to properly expand in embryos homozygous for either of these genes. In addition to these durable homozygous phenotypes, we also demonstrated recoverable delays in ptf1a-expressing pancreatic progenitor expansion in rpl23a(hi2582) and rpl6(hi3655b) heterozygotes. Disruptions in ribosome assembly are generally understood to initiate a p53-dependent cellular stress response. However, concomitant p53 knockdown was unable to rescue normal pancreatic progenitor expansion in either rpl23a(hi2582) or rpl6(hi3655b) mutant embryos, suggesting required and p53-independent roles for rpl23a and rpl6 in pancreas development.

  2. Characterization of mesonephric development and regeneration using transgenic zebrafish

    PubMed Central

    Zhou, Weibin; Boucher, Rudrick C.; Bollig, Frank; Englert, Christoph

    2010-01-01

    The zebrafish is a valuable vertebrate model for kidney research. The majority of previous studies focused on the zebrafish pronephros, which comprises only two nephrons and is structurally simpler than the mesonephros of adult fish and the metanephros of mammals. To evaluate the zebrafish system for more complex studies of kidney development and regeneration, we investigated the development and postinjury regeneration of the mesonephros in adult zebrafish. Utilizing two transgenic zebrafish lines (wt1b::GFP and pod::NTR-mCherry), we characterized the developmental stages of individual mesonephric nephrons and the temporal-spatial pattern of mesonephrogenesis. We found that mesonephrogenesis continues throughout the life of zebrafish, with a rapid growth phase during the juvenile period and a slower phase in adulthood such that the total nephron number of juvenile and adult fish linearly correlates with body mass. Following gentamicin-induced renal injury, the zebrafish mesonephros can undergo de novo regeneration of mesonephric nephrons, a process known as neonephrogenesis. We found that wt1b expression was induced in individually dispersed cells in the mesonephric interstitium as early as 48 h following injury. These wt1b-expressing cells formed aggregates by 72–96 h following injury which proceeded to form nephrons. This suggests that wt1b may serve as an early marker of fated renal progenitor cells. The synchronous nature of regenerative neonephrogenesis suggests that this process may be useful for studies of nephron development. PMID:20810610

  3. Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors.

    PubMed

    Lam, Enid Yi Ni; Chau, Jackie Y M; Kalev-Zylinska, Maggie L; Fountaine, Timothy M; Mead, R Scott; Hall, Christopher J; Crosier, Philip S; Crosier, Kathryn E; Flores, Maria Vega

    2009-02-05

    The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein-labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter-transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

  4. The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development.

    PubMed

    Bedell, Victoria M; Person, Anthony D; Larson, Jon D; McLoon, Anna; Balciunas, Darius; Clark, Karl J; Neff, Kevin I; Nelson, Katie E; Bill, Brent R; Schimmenti, Lisa A; Beiraghi, Soraya; Ekker, Stephen C

    2012-02-01

    The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

  5. Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish, Mice and Humans

    PubMed Central

    Kaufeld, Jessica; Miller, Emily; Tossidou, Irini; Englert, Christoph; Bollig, Frank; Staggs, Lynne; Roberts, Ian S. D.; Park, Joon-Keun; Haller, Hermann; Schiffer, Mario

    2010-01-01

    Background Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration. Methods and Findings We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus. Conclusions Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development. PMID:20838616

  6. Stem Cell Differentiation Stage Factors from Zebrafish Embryo: A Novel Strategy to Modulate the Fate of Normal and Pathological Human (Stem) Cells

    PubMed Central

    Biava, Pier M.; Canaider, Silvia; Facchin, Federica; Bianconi, Eva; Ljungberg, Liza; Rotilio, Domenico; Burigana, Fabio; Ventura, Carlo

    2015-01-01

    In spite of the growing body of evidence on the biology of the Zebrafish embryo and stem cells, including the use of Stem Cell Differentiation Stage Factors (SCDSFs) taken from Zebrafish embryo to impact cancer cell dynamics, comparatively little is known about the possibility to use these factors to modulate the homeostasis of normal human stem cells or to modulate the behavior of cells involved in different pathological conditions. In the present review we recall in a synthetic way the most important researches about the use of SCDSFs in reprogramming cancer cells and in modulating the high speed of multiplication of keratinocytes which is characteristic of some pathological diseases like psoriasis. Moreover we add here the results about the capability of SCDSFs in modulating the homeostasis of human adipose-derived stem cells (hASCs) isolated from a fat tissue obtained with a novel-non enzymatic method and device. In addition we report the data not yet published about a first protein analysis of the SCDSFs and about their role in a pathological condition like neurodegeneration. PMID:26201607

  7. Stem Cell Differentiation Stage Factors from Zebrafish Embryo: A Novel Strategy to Modulate the Fate of Normal and Pathological Human (Stem) Cells.

    PubMed

    Biava, Pier M; Canaider, Silvia; Facchin, Federica; Bianconi, Eva; Ljungberg, Liza; Rotilio, Domenico; Burigana, Fabio; Ventura, Carlo

    2015-01-01

    In spite of the growing body of evidence on the biology of the Zebrafish embryo and stem cells, including the use of Stem Cell Differentiation Stage Factors (SCDSFs) taken from Zebrafish embryo to impact cancer cell dynamics, comparatively little is known about the possibility to use these factors to modulate the homeostasis of normal human stem cells or to modulate the behavior of cells involved in different pathological conditions. In the present review we recall in a synthetic way the most important researches about the use of SCDSFs in reprogramming cancer cells and in modulating the high speed of multiplication of keratinocytes which is characteristic of some pathological diseases like psoriasis. Moreover we add here the results about the capability of SCDSFs in modulating the homeostasis of human adiposederived stem cells (hASCs) isolated from a fat tissue obtained with a novel-non enzymatic method and device. In addition we report the data not yet published about a first protein analysis of the SCDSFs and about their role in a pathological condition like neurodegeneration.

  8. Epithelial cell fate in the nephron tubule is mediated by the ETS transcription factors etv5a and etv4 during zebrafish kidney development.

    PubMed

    Marra, Amanda N; Wingert, Rebecca A

    2016-03-15

    Kidney development requires the differentiation and organization of discrete nephron epithelial lineages, yet the genetic and molecular pathways involved in these events remain poorly understood. The embryonic zebrafish kidney, or pronephros, provides a simple and useful model to study nephrogenesis. The pronephros is primarily comprised of two types of epithelial cells: transportive and multiciliated cells (MCCs). Transportive cells occupy distinct tubule segments and are characterized by the expression of various solute transporters, while MCCs function in fluid propulsion and are dispersed in a "salt-and-pepper" fashion within the tubule. Epithelial cell identity is reliant on interplay between the Notch signaling pathway and retinoic acid (RA) signaling, where RA promotes MCC fate by inhibiting Notch activity in renal progenitors, while Notch acts downstream to trigger transportive cell formation and block adoption of an MCC identity. Previous research has shown that the transcription factor ets variant 5a (etv5a), and its closely related ETS family members, are required for ciliogenesis in other zebrafish tissues. Here, we mapped etv5a expression to renal progenitors that occupy domains where MCCs later emerge. Thus, we hypothesized that etv5a is required for normal development of MCCs in the nephron. etv5a loss of function caused a decline of MCC number as indicated by the reduced frequency of cells that expressed the MCC-specific markers outer dense fiber of sperm tails 3b (odf3b) and centrin 4 (cetn4), where rescue experiments partially restored MCC incidence. Interestingly, deficiency of ets variant 4 (etv4), a related gene that is broadly expressed in the posterior mesoderm during somitogenesis stages, also led to reduced MCC numbers, which were further reduced by dual etv5a/4 deficiency, suggesting that both of these ETS factors are essential for MCC formation and that they also might have redundant activities. In epistatic studies, exogenous RA

  9. A genomic region encompassing a newly identified exon provides enhancing activity sufficient for normal myo7aa expression in zebrafish sensory hair cells.

    PubMed

    Ernest, Sylvain; Rosa, Frédéric M

    2015-09-01

    MYO7A is an unconventional myosin involved in the structural organization of hair bundles at the apex of sensory hair cells (SHCs) where it serves mechanotransduction in the process of hearing and balance. Mutations of MYO7A are responsible for abnormal shaping of hair bundles, resulting in human deafness and murine deafness/circling behavior. Myo7aa, expressed in SHCs of the inner ear and lateral line of zebrafish, causes circling behavior and abnormal hair cell function when deficient in mariner mutant. This work identifies a new hair cell-specific enhancer, highly conserved between species, located within Intron 2-3 of zebrafish myosin 7a (myo7aa) gene. This enhancer is contained within a 761-bp DNA fragment that encompasses a newly identified Exon of myo7aa and whose activity does not depend on orientation. Compensation of mariner mutation by expression of mCherry-Myo7aa fusion protein under the control of this 761-bp DNA fragment results in recovery of balance, normal hair bundle shape and restored hair cell function. Two smaller adjacent fragments (344-bp and 431-bp), extracted from the 761-bp fragment, both show hair cell-specific enhancing activity, with apparently reduced intensity and coverage. These data should help understand the role of Myo7aa in sensory hair cell differentiation and function. They provide tools to decipher how myo7aa gene is expressed and regulated in SHCs by allowing the identification of potential transcription factors involved in this process. The discovered enhancer could represent a new target for the identification of deafness-causing mutations affecting human MYO7A.

  10. Prostaglandin signaling regulates nephron segment patterning of renal progenitors during zebrafish kidney development

    PubMed Central

    Poureetezadi, Shahram Jevin; Cheng, Christina N; Chambers, Joseph M; Drummond, Bridgette E; Wingert, Rebecca A

    2016-01-01

    Kidney formation involves patterning events that induce renal progenitors to form nephrons with an intricate composition of multiple segments. Here, we performed a chemical genetic screen using zebrafish and discovered that prostaglandins, lipid mediators involved in many physiological functions, influenced pronephros segmentation. Modulating levels of prostaglandin E2 (PGE2) or PGB2 restricted distal segment formation and expanded a proximal segment lineage. Perturbation of prostaglandin synthesis by manipulating Cox1 or Cox2 activity altered distal segment formation and was rescued by exogenous PGE2. Disruption of the PGE2 receptors Ptger2a and Ptger4a similarly affected the distal segments. Further, changes in Cox activity or PGE2 levels affected expression of the transcription factors irx3b and sim1a that mitigate pronephros segment patterning. These findings show for the first time that PGE2 is a regulator of nephron formation in the zebrafish embryonic kidney, thus revealing that prostaglandin signaling may have implications for renal birth defects and other diseases. DOI: http://dx.doi.org/10.7554/eLife.17551.001 PMID:27996936

  11. Genome-wide identification of suitable zebrafish Danio rerio reference genes for normalization of gene expression data by RT-qPCR.

    PubMed

    Xu, H; Li, C; Zeng, Q; Agrawal, I; Zhu, X; Gong, Z

    2016-06-01

    In this study, to systematically identify the most stably expressed genes for internal reference in zebrafish Danio rerio investigations, 37 D. rerio transcriptomic datasets (both RNA sequencing and microarray data) were collected from gene expression omnibus (GEO) database and unpublished data, and gene expression variations were analysed under three experimental conditions: tissue types, developmental stages and chemical treatments. Forty-four putative candidate genes were identified with the c.v. <0·2 from all datasets. Following clustering into different functional groups, 21 genes, in addition to four conventional housekeeping genes (eef1a1l1, b2m, hrpt1l and actb1), were selected from different functional groups for further quantitative real-time (qrt-)PCR validation using 25 RNA samples from different adult tissues, developmental stages and chemical treatments. The qrt-PCR data were then analysed using the statistical algorithm refFinder for gene expression stability. Several new candidate genes showed better expression stability than the conventional housekeeping genes in all three categories. It was found that sep15 and metap1 were the top two stable genes for tissue types, ube2a and tmem50a the top two for different developmental stages, and rpl13a and rp1p0 the top two for chemical treatments. Thus, based on the extensive transcriptomic analyses and qrt-PCR validation, these new reference genes are recommended for normalization of D. rerio qrt-PCR data respectively for the three different experimental conditions. © 2016 The Fisheries Society of the British Isles.

  12. Zebrafish nephrogenesis is regulated by interactions between retinoic acid, mecom, and Notch signaling

    PubMed Central

    Li, Yue; Cheng, Christina N.; Verdun, Valerie A.; Wingert, Rebecca A.

    2014-01-01

    The zebrafish pronephros provides a conserved model to study kidney development, in particular to delineate the poorly understood processes of how nephron segment pattern and cell type choice are established. Zebrafish nephrons are divided into distinct epithelial regions that include a series of proximal and distal tubule segments, which are comprised of intercalated transporting epithelial cells and multiciliated cells (MCC). Previous studies have shown that retinoic acid (RA) regionalizes the renal progenitor field into proximal and distal domains and that Notch signaling later represses MCC differentiation, but further understanding of these pathways has remained unknown. The transcription factor mecom (mds1/evi1 complex) is broadly expressed in renal progenitors, and then subsequently marks the distal tubule. Here, we show that mecom is necessary to form the distal tubule and to restrict both proximal tubule formation and MCC fate choice. We found that mecom and RA have opposing roles in patterning discrete proximal and distal segments. Further, we discovered that RA is required for MCC formation, and that one mechanism by which RA promotes MCC fate choice is to inhibit mecom. Next, we determined the epistatic relationship between mecom and Notch signaling, which limits MCC fate choice by lateral inhibition. Abrogation of Notch signaling with the y-secretase inhibitor DAPT revealed that Notch and mecom did not have additive effects in blocking MCC formation, suggesting that they function in the same pathway. Ectopic expression of the Notch signaling effector, Notch intracellular domain (NICD), rescued the expansion of MCCs in mecom morphants, indicating that mecom acts upstream to induce Notch signaling. These findings suggest a model in which mecom and RA arbitrate proximodistal segment domains, while MCC fate is modulated by a complex interplay in which RA inhibition of mecom, and mecom promotion of Notch, titrates MCC number. Taken together, our studies

  13. Zebrafish nephrogenesis involves dynamic spatiotemporal expression changes in renal progenitors and essential signals from retinoic acid and irx3b

    PubMed Central

    Wingert, Rebecca A.; Davidson, Alan J.

    2013-01-01

    Kidney nephrons are comprised of proximal and distal tubule segments that perform unique roles in excretion. The developmental pathways that establish nephron segment identities from renal progenitors are poorly understood. Here, we used the zebrafish pronephros to study nephron segmentation. We found that zebrafish nephron progenitors undergo elaborate spatiotemporal expression changes of many genes before adopting a segment fate. Initially, two domains of nephron progenitors are established, then are subdivided and demarcate individual nephron segments. Using genetic and chemical genetic models of retinoic acid (RA) deficiency, we discovered that RA modulates rostral progenitor formation. To delineate downstream pathways, we knocked down the irx3b transcription factor and found it regulates proximal tubule segment size and distal segment differentiation. Our results suggest a model whereby RA patterns the early field of nephron progenitors, with subsequent factors like irx3b acting to refine later progenitor subdomains and ensure activation of segment-specific gene programs. PMID:21761484

  14. Functional aging and gradual senescence in zebrafish.

    PubMed

    Kishi, Shuji

    2004-06-01

    Zebrafish (Danio rerio) has been recognized as a powerful model for genetic studies in developmental biology. Recently, the zebrafish system also has given insights into several human diseases such as neurodegenerative, hematopoietic, and cardiovascular disease, and cancer. Because aging processes affect these and various other human disorders, it is important to compare zebrafish and mammalian senescence. However, the aging process of zebrafish remains largely unexplored, and little is known about functional aging and senescence in zebrafish. In our initial studies to assess aging phenotypes in zebrafish, we have identified several potential aging biomarkers in an ongoing search for suitable ones on zebrafish aging. In aging zebrafish, we detected senescence-associated beta-galactosidase activity in skin and oxidized protein accumulation in muscle. On the other hand, we did not observe lipofuscin granules (aging pigments), which accumulate in postmitotic cells, in muscle of zebrafish with advancing age. Consistently, there were continuously proliferating myocytes that incorporated BrdU in muscle tissues of the aged fish. Moreover, we demonstrated that zebrafish have constitutively abundant telomerase activity in adult somatic tissues implicating unlimited replicative ability of cells throughout their lives. Although some stress-associated markers are upregulated and minor histological changes are observed during the aging process of zebrafish, our studies together with other evidence of remarkable reproductive and regenerative abilities suggest that zebrafish show very gradual senescence. By using those biological and biochemical aging markers already characterized in normal zebrafish, transgenic fish analyses and genetic mutant fish screens can be readily performed. These efforts will help to elucidate the role and molecular mechanisms of common or different pathways of aging among vertebrates from fish to humans and also will contribute to the discovery of

  15. In vitro effect of mercuric chloride and sodium selenite on chemiluminescent response of pronephros cells isolated from Tilapia, oreochromis aureus

    SciTech Connect

    Low, K.W.; Sin, Y.M.

    1995-12-01

    Phagocytosis is a basic immunological function of mononuclear phagocytes and polymorphonuclear leukocytes. This process is a major defence mechanism in fish which involves recognition and killing of pathogenic microorganisms. It has been reported that phagocytic cells consume more oxygen and release several reactive oxygen species (ROS) during phagocytosis. This {open_quote}respiratory burst{close_quote} was first quantified by measuring the chemiluminescence (CL) emitted from human polymorphonuclear leukocytes and later in fish phagocytes. The oxygen intermediates responsible for this CL reaction include O{sub 2}{sup {minus}}, {center_dot}OH and H{sub 2}O{sub 2} which are also the major bactericidal agents in phagocytes{prime} oxygen-dependent killing process. Therefore, CL response can be used as an indicator of phagocytosis. This study is designed to examine the individual effects of mercury and selenium and also their possible interaction on CL response of fish pronephros phagocytes, because a defect in phagocytosis may predispose fish to diseases. 25 refs., 3 tabs.

  16. Edwardsiellosis Caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio

    PubMed Central

    Hawke, John P.; Kent, Michael; Rogge, Matt; Baumgartner, Wes; Wiles, Judy; Shelley, Johnny; Savolainen, L. Christine; Wagner, Robert; Murray, Katy; Peterson, Tracy S.

    2014-01-01

    We report the first cases of Edwardsiella ictaluri causing epizootics in laboratory populations of Zebrafish Danio rerio. Edwardsiella ictaluri is primarily recognized as a disease of catfish species and is known to cause an economically important bacterial disease of farm-raised catfish in the USA and abroad; however, it has been isolated on occasion from 10 other genera of nonictalurid fishes. We isolated E. ictaluri from moribund Zebrafish held in quarantine at two different universities in two states and from a research facility in a third state between February 23 and December 6, 2011. Edwardsiellosis in Zebrafish can be described as a severe systemic disease characterized by tissue necrosis and the presence of large numbers of extracellular and intracellular bacteria, often within macrophages. The kidneys (pronephros and mesonephros), spleen, nares, and forebrain were the most commonly and severely affected tissues. In outbreaks, mortality was acute and numerous fish died over a 1–2 week period. Mortality continued until the majority of the population was lost, at which time the remaining fish were euthanized. In addition to these cases, four cultures of bacteria isolated from Zebrafish by another diagnostic laboratory were submitted to the Louisiana Aquatic Diagnostic Laboratory for identification and were confirmed as E. ictaluri. In total, eight cultures of E. ictaluri from Zebrafish from Louisiana, Massachusetts, Pennsylvania, and Florida were identified. The isolates were confirmed as E. ictaluri by biochemical phenotype, API 20E (bioMérieux), and amplification and sequencing of a portion of the 16S rRNA gene. Edwardsiella ictaluri isolates from Zebrafish are believed to comprise a unique group and were differentiated from catfish isolates by exhibiting weaker motility, autoaggregation in broth, a different plasmid profile (two plasmids of 4.0 and 3.5 kb), a different API 20E code (4204000), and lack of lipopolysaccharide recognition with Mab Ed9

  17. Quantifying Aggressive Behavior in Zebrafish.

    PubMed

    Teles, Magda C; Oliveira, Rui F

    2016-01-01

    Aggression is a complex behavior that influences social relationships and can be seen as adaptive or maladaptive depending on the context and intensity of expression. A model organism suitable for genetic dissection of the underlying neural mechanisms of aggressive behavior is still needed. Zebrafish has already proven to be a powerful vertebrate model organism for the study of normal and pathological brain function. Despite the fact that zebrafish is a gregarious species that forms shoals, when allowed to interact in pairs, both males and females express aggressive behavior and establish dominance hierarchies. Here, we describe two protocols that can be used to quantify aggressive behavior in zebrafish, using two different paradigms: (1) staged fights between real opponents and (2) mirror-elicited fights. We also discuss the methodology for the behavior analysis, the expected results for both paradigms, and the advantages and disadvantages of each paradigm in face of the specific goals of the study.

  18. Identification and expression of soul/p22HBP genes in zebrafish.

    PubMed

    Fortunato, Antonio Emidio; Langellotto, Fernanda; Sordino, Paolo

    2011-01-01

    The SOUL/p22HBP family is an evolutionarily ancient group of heme binding proteins with a main function as cytosolic buffer against tetrapyrrole accumulation. Structural and biochemical evidence suggest specialized roles in blood formation, necrotic cell death and chemotaxis. To date, nothing is known about the precise activity and expression patterns of this class of heme binding proteins during development. The zebrafish genome possesses five soul genes belonging to two subgroups, and no p22HBP orthologous gene. Here, spatial and temporal expression patterns are reported for zebrafish soul1, soul2 and soul4 genes. All three soul genes are maternally transcribed, and their zygotic expression takes place in unique (heart, pharynx, yolk syncytial layer, brain, eyes, lateral line) and overlapping (pronephros, pituitary gland, olfactory and otic vesicle) regions of the zebrafish embryo. Our study constitutes the first detailed analysis of soul gene expression in metazoan development, and provides the basis to understand the genetics of tetrapyrrole metabolism in a wide range of embryonic processes.

  19. Evidence of a role of inositol polyphosphate 5-phosphatase INPP5E in cilia formation in zebrafish.

    PubMed

    Luo, Na; Lu, Jingping; Sun, Yang

    2012-12-15

    Inositol phosphatases are important regulators of cell signaling and membrane trafficking. Mutations in inositol polyphosphate 5-phosphatase, INPP5E, have been identified in Joubert syndrome, a rare congenital disorder characterized by midbrain malformation, retinitis pigmentosa, renal cysts, and polydactyly. Previous studies have implicated primary cilia abnormalities in Joubert syndrome, yet the role of INPP5E in cilia formation is not well understood. In this study, we examined the function of INPP5E in cilia development in zebrafish. Using specific antisense morpholino oligonucleotides to knockdown Inpp5e expression, we observed phenotypes of microphthalmia, pronephros cysts, pericardial effusion, and left-right body axis asymmetry. The Inpp5e morphant zebrafish exhibited shortened and decreased cilia formation in the Kupffer's vesicle and pronephric ducts as compared to controls. Epinephrine-stimulated melanosome trafficking was delayed in the Inpp5e zebrafish morphants. Expression of human INPP5E expression rescued the phenotypic defects in the Inpp5e morphants. Taken together, we showed that INPP5E is critical for the cilia development in zebrafish.

  20. Visualization of stochastic Ca2+ signals in the formed somites during the early segmentation period in intact, normally developing zebrafish embryos.

    PubMed

    Leung, Christina F; Miller, Andrew L; Korzh, Vladimir; Chong, Shang-Wei; Sleptsova-Freidrich, Inna; Webb, Sarah E

    2009-09-01

    Localized Ca(2+) signals were consistently visualized in the formed somites of intact zebrafish embryos during the early segmentation period. Unlike the regular process of somitogenesis, these signals were stochastic in nature with respect to time and location. They did, however, occur predominantly at the medial and lateral boundaries within the formed somites. Embryos were treated with modulators of [Ca(2+)](i) to explore the signal generation mechanism and possible developmental function of the stochastic transients. Blocking elements in the phosphoinositol pathway eliminated the stochastic signals but had no obvious effect, stochastic or otherwise, on the formed somites. Such treatments did, however, result in the subsequently formed somites being longer in the mediolateral dimension. Targeted uncaging of buffer (diazo-2) or Ca(2+) (NP-ethyleneglycoltetraacetic acid [EGTA]) in the presomitic mesoderm, resulted in a regular mediolateral lengthening and shortening, respectively, of subsequently formed somites. These data suggest a requirement for IP(3) receptor-mediated Ca(2+) release during convergence cell movements in the presomitic mesoderm, which appears to have a distinct function from that of the IP(3) receptor-mediated stochastic Ca(2+) signaling in the formed somites.

  1. Duplicate sfrp1 genes in zebrafish: sfrp1a is dynamically expressed in the developing central nervous system, gut and lateral line.

    PubMed

    Pézeron, Guillaume; Anselme, Isabelle; Laplante, Mary; Ellingsen, Staale; Becker, Thomas S; Rosa, Frédéric M; Charnay, Patrick; Schneider-Maunoury, Sylvie; Mourrain, Philippe; Ghislain, Julien

    2006-10-01

    The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.

  2. Renin expression in developing zebrafish is associated with angiogenesis and requires the Notch pathway and endothelium

    PubMed Central

    Mullins, Linda J.; Verdon, Rachel F.; MacRae, Calum A.; Mullins, John J.

    2015-01-01

    Although renin is a critical regulatory enzyme of the cardiovascular system, its roles in organogenesis and the establishment of cardiovascular homeostasis remain unclear. Mammalian renin-expressing cells are widespread in embryonic kidneys but are highly restricted, specialized endocrine cells in adults. With a functional pronephros, embryonic zebrafish are ideal for delineating the developmental functions of renin-expressing cells and the mechanisms governing renin transcription. Larval zebrafish renin expression originates in the mural cells of the juxtaglomerular anterior mesenteric artery and subsequently at extrarenal sites. The role of renin was determined by assessing responses to renin-angiotensin system blockade, salinity variation, and renal perfusion ablation. Renin expression did not respond to renal flow ablation but was modulated by inhibition of angiotensin-converting enzyme and altered salinity. Our data in larval fish are consistent with conservation of renin's physiological functions. Using transgenic renin reporter fish, with mindbomb and cloche mutants, we show that Notch signaling and the endothelium are essential for developmental renin expression. After inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts. Arising from separate lineages, but relying on mutual interplay with endothelial cells, renin-expressing cells are among the earliest mural cells observed in larval fish, performing both endocrine and paracrine functions. PMID:26202224

  3. Hearing Assessment in Zebrafish During the First Week Postfertilization

    PubMed Central

    Yao, Qi; DeSmidt, Alexandra A.; Tekin, Mustafa; Liu, Xuezhong

    2016-01-01

    Abstract The zebrafish (Danio rerio) is a valuable vertebrate model for human hearing disorders because of many advantages in genetics, embryology, and in vivo visualization. In this study, we investigated auditory function of zebrafish during the first week postfertilization using microphonic potential recording. Extracellular microphonic potentials were recorded from hair cells in the inner ear of wild-type AB and transgenic Et(krt4:GFP)sqet4 zebrafish at 3, 5, and 7 days postfertilization in response to 20, 50, 100, 200, 300, and 400-Hz acoustic stimulation. We found that microphonic threshold significantly decreased with age in zebrafish. However, there was no significant difference of microphonic responses between wild-type and transgenic zebrafish, indicating that the transgenic zebrafish have normal hearing like wild-type zebrafish. In addition, we observed that microphonic threshold did not change with the recording electrode location. Furthermore, microphonic threshold increased significantly at all tested stimulus frequencies after displacement of the saccular otolith but only increased at low frequencies after displacement of the utricular otolith, showing that the saccule rather than the utricle plays the major role in larval zebrafish hearing. These results enhance our knowledge of early development of auditory function in zebrafish and the factors affecting hearing assessment with microphonic potential recording. PMID:26982161

  4. Hearing Assessment in Zebrafish During the First Week Postfertilization.

    PubMed

    Yao, Qi; DeSmidt, Alexandra A; Tekin, Mustafa; Liu, Xuezhong; Lu, Zhongmin

    2016-04-01

    The zebrafish (Danio rerio) is a valuable vertebrate model for human hearing disorders because of many advantages in genetics, embryology, and in vivo visualization. In this study, we investigated auditory function of zebrafish during the first week postfertilization using microphonic potential recording. Extracellular microphonic potentials were recorded from hair cells in the inner ear of wild-type AB and transgenic Et(krt4:GFP)(sqet4) zebrafish at 3, 5, and 7 days postfertilization in response to 20, 50, 100, 200, 300, and 400-Hz acoustic stimulation. We found that microphonic threshold significantly decreased with age in zebrafish. However, there was no significant difference of microphonic responses between wild-type and transgenic zebrafish, indicating that the transgenic zebrafish have normal hearing like wild-type zebrafish. In addition, we observed that microphonic threshold did not change with the recording electrode location. Furthermore, microphonic threshold increased significantly at all tested stimulus frequencies after displacement of the saccular otolith but only increased at low frequencies after displacement of the utricular otolith, showing that the saccule rather than the utricle plays the major role in larval zebrafish hearing. These results enhance our knowledge of early development of auditory function in zebrafish and the factors affecting hearing assessment with microphonic potential recording.

  5. Modulatory effect of metal ions on the immune response of fish: in vivo and in vitro influence of MnCl sub 2 on NK activity of carp pronephros cells

    SciTech Connect

    Ghanmi, Z.; Rouabhia, M.; Alifuddin, M.; Troutaud, D.; Deschaux, P. )

    1990-12-01

    The in vivo and in vitro influence of MnCl{sub 2} on carp pronephros cells was investigated. Increased cytotoxicity against both YAC-1 and P 815 target cells was observed following an intraperitoneal injection of 40, 80, or 120 micrograms MnCl{sub 2}/g body wt administrated 24 hr prior to the in vitro {sup 51}Cr release assay. Similarly, in vitro treatment of carp pronephros cells, at a final concentration of 60 micrograms/culture, resulted in an increase of NK cell activity in both YAC-1 and P 815 target cell lines. However, a significant decrease in this activity was shown with lower doses of MnCl{sub 2} (40 and 20 micrograms/culture).

  6. Zebrafish teratogenicity testing.

    PubMed

    Brannen, Kimberly C; Charlap, Jeffrey H; Lewis, Elise M

    2013-01-01

    As a model for teratogenicity research, zebrafish are gaining popularity and creditability. Zebrafish embryos have been proven to be a highly valuable tool in genetics and developmental biology research and have advanced our understanding of a number of known developmental toxicants. It has yet to be determined conclusively how reliably a zebrafish embryo screening assay predicts what will happen in mammalian models, but results from initial assessments have been encouraging. Here we have presented procedures for the basic care of a zebrafish colony to support embryo production, embryo collection and culturing, and teratogenicity experiments.

  7. Stressing Zebrafish for Behavioral Genetics

    PubMed Central

    Clark, Karl J.; Boczek, Nicole J.; Ekker, Stephen C.

    2012-01-01

    Synopsis The stress response is a normal reaction to a real or perceived threat. However, stress response systems that are overwhelmed or out of balance can increase both the incidence and severity of diseases including addiction and mood and anxiety disorders. Using an animal model with both genetic diversity and large family size can help discover the specific genetic and environmental contributions to these behavioral diseases. The stress response has been studied extensively in teleosts because of their importance in food production. The zebrafish (Danio rerio) is a major model organism with a strong record for use in developmental biology, genetic screening, and genomic studies. More recently, the stress response of larval and adult zebrafish has been documented. High-throughput automated tracking systems make possible behavioral readouts of the stress response in zebrafish. This non-invasive measure of the stress response can be combined with mutagenesis methods to dissect the genes involved in complex stress response behaviors in vertebrates. Understanding the genetic and epigenetic basis for the stress response in vertebrates will help to develop advanced screening and therapies for stress-aggravated diseases like addiction and mood and anxiety disorders. PMID:21615261

  8. Zebrafish Discoveries in Cancer Epigenetics

    PubMed Central

    Chernyavskaya, Yelena; Kent, Brandon

    2017-01-01

    The cancer epigenome is fundamentally different than that of normal cells. How these differences arise in and contribute to carcinogenesis is not known, and studies using model organisms such as zebrafish provide an opportunity to address these important questions. Modifications of histones and DNA comprise the complex epigenome, and these influence chromatin structure, genome stability and gene expression, all of which are fundamental to the cellular changes that cause cancer. The cancer genome atlas covers the wide spectrum of genetic changes associated with nearly every cancer type, however, this catalog is currently unidimensional. As the pattern of epigenetic marks and chromatin structure in cancer cells is described and overlaid on the mutational landscape, the map of the cancer genome becomes multi-dimensional and highly complex. Two major questions remain in the field: (1) how the epigenome becomes repatterned in cancer and (2) which of these changes are cancer-causing. Zebrafish provide a tractable in vivo system to monitor the epigenome during transformation and to identify epigenetic drivers of cancer. In this chapter, we review principles of cancer epigenetics and discuss recent work using zebrafish whereby epigenetic modifiers were established as cancer driver genes, thus providing novel insights into the mechanisms of epigenetic reprogramming in cancer. PMID:27165354

  9. The Zebrafish Secretome

    PubMed Central

    2008-01-01

    Abstract The secretome is a functionally rich proteome subset, including cellular membrane and extracellular proteins processed through the secretory pathway. In this study, Danio rerio and Homo sapiens RefSeq proteins were analyzed with SignalP, TargetP, Phobius, and pTarget algorithms. About 16.5% of the zebrafish proteome and 17.0% of the human proteome possessed predicted N-terminal signal sequences. Nearly half of these proteins were subsequently classified as soluble, as they lacked predicted transmembrane domains. The soluble proteins were further subclassified, predicting 1345 (3.8%) zebrafish and 1207 (3.2%) human proteins as extracellular. Comparison of the zebrafish and human soluble secretome proteins identified 372 as orthologs, on the basis of reciprocal BLAST best hits. The computational characterization of the zebrafish proteins found many more members of the secretome than annotated in the SwissProt database. Only 180 of the 2078 zebrafish SwissProt protein entries, and 995 of the 19,294 human SwissProt protein entries were annotated with secreted protein locales. A specific investigation of the fibroblast growth factor and matrix metalloproteinase (MMP) protein families confirmed the prediction data and generated annotation of three additional putative MMP zebrafish proteins. This study presents the first known published description of the zebrafish secretome since the completion of the zebrafish genome sequencing project. PMID:18554177

  10. Molecular psychiatry of zebrafish

    PubMed Central

    Stewart, Adam Michael; Ullmann, Jeremy F.P.; Norton, William H.J.; Brennan, Caroline H.; Parker, Matthew O.; Gerlai, Robert; Kalueff, Allan V.

    2014-01-01

    Due to their well-characterized neural development and high genetic homology to mammals, zebrafish (Danio rerio) have emerged as a powerful model organism in the field of biological psychiatry. Here, we discuss the molecular psychiatry of zebrafish, and its implications for translational neuroscience research and modeling CNS disorders. In particular, we outline recent genetic and technological developments allowing for in-vivo examinations, high-throughput screening and whole-brain analyses in larval and adult zebrafish. We also summarize the application of these molecular techniques to the understanding of neuropsychiatric disease, outlining the potential of zebrafish for modeling complex brain disorders, including attention-deficit/hyperactivity disorder (ADHD), aggression, post-traumatic stress and substance abuse. Critically evaluating the advantages and limitations of larval and adult fish tests, we suggest that zebrafish models become a rapidly emerging new field in modern biological psychiatry research. PMID:25349164

  11. Electroporation of adult zebrafish.

    PubMed

    Rao, N Madhusudhana; Rambabu, K Murali; Rao, S Harinarayana

    2008-01-01

    We generated transient transgenic zebrafish by applying electrical pulses subsequent to injection of DNA into muscle tissue of 3-6-month old adult zebrafish. Electroporation parameters, such as number of pulses, voltage, and amount of plasmid DNA, were optimized and found that 6 pulses of 40 V/cm at 15 mug/fish increased the luciferase expression by 10-fold compared with those in controls. By measuring the expression of luciferase, in vivo by electroporation in adult zebrafish and in vitro using fish cell line (Xiphophorus xiphidium A2 cells), the strength of three promoters (CMV, human EF-1alpha, and Xenopus EF-1alpha) was compared. Subsequent to electroporation after injecting DNA in the mid region of zebrafish, expression of green fluorescent protein was found far away from the site of injection in the head and the tail sections. Thus, electroporation in adult zebrafish provides a rapid way of testing the behavior of gene sequences in the whole organism.

  12. Application of complementary luminescent and fluorescent imaging techniques to visualize nuclear and cytoplasmic Ca²⁺ signalling during the in vivo differentiation of slow muscle cells in zebrafish embryos under normal and dystrophic conditions.

    PubMed

    Webb, Sarah E; Cheung, Chris C Y; Chan, Ching Man; Love, Donald R; Miller, Andrew L

    2012-01-01

    1. Evidence is accumulating for a role for Ca²⁺ signalling in the differentiation and development of embryonic skeletal muscle. 2. Imaging of intact, normally developing transgenic zebrafish that express the protein component of the Ca²⁺-sensitive complex aequorin, specifically in skeletal muscle, show that two distinct periods of spontaneous synchronised Ca²⁺ transients occur in the trunk: one at approximately 17.5-19.5 h post-fertilization (h.p.f.; termed signalling period SP1) and the other after approximately 23 h.p.f. (termed SP2). These periods of intense Ca²⁺ signalling activity are separated by a quiet period. 3. Higher-resolution confocal imaging of embryos loaded with the fluorescent Ca²⁺ reporter calcium green-1 dextran shows that the Ca²⁺ signals are generated almost exclusively in the slow muscle cells, the first muscle cells to differentiate, with distinct nuclear and cytoplasmic components. 4. Here, we show that coincidental with the SP1 Ca²⁺ signals, dystrophin becomes localized to the vertical myoseptae of the myotome. Introduction of a dmd morpholino (dmd-MO) resulted in no dystrophin being expressed in the vertical myoseptae, as well as a disruption of myotome morphology and sarcomere organization. In addition, the Ca²⁺ signalling signatures of dmd-MO-injected embryos or homozygous sapje mutant embryos were abnormal such that the frequency, amplitude and timing of the Ca²⁺ signals were altered compared with controls. 5. Our new data suggest that, in addition to a structural role, dystrophin may function in the regulation of [Ca²⁺](i) during the early stages of slow muscle cell differentiation when the Ca²⁺ signals generated in these cells coincide with the first spontaneous contractions of the trunk.

  13. A reverse genetic screen in the zebrafish identifies crb2b as a regulator of the glomerular filtration barrier.

    PubMed

    Ebarasi, Lwaki; He, Liqun; Hultenby, Kjell; Takemoto, Minoru; Betsholtz, Christer; Tryggvason, Karl; Majumdar, Arindam

    2009-10-01

    The glomerular filtration barrier is necessary for the selective passage of low molecular weight waste products and the retention of blood plasma proteins. Damage to the filter results in proteinuria. The filtration barrier is the major pathogenic site in almost all glomerular diseases and its study is therefore of clinical significance. We have taken advantage of the zebrafish pronephros as a system for studying glomerular filtration. In order to identify new regulators of filtration barrier assembly, we have performed a reverse genetic screen in the zebrafish testing a group of genes which are enriched in their expression within the mammalian glomerulus. In this novel screen, we have coupled gene knockdown using morpholinos with a physiological glomerular dye filtration assay to test for selective glomerular permeability in living zebrafish larvae. Screening 20 genes resulted in the identification of ralgps1, rapgef2, rabgef1, and crb2b. The crumbs (crb) genes encode a family of evolutionarily conserved proteins important for apical-basal polarity within epithelia. The crb2b gene is expressed in zebrafish podocytes. Electron microscopic analysis of crb2b morphants reveals a gross disorganization of podocyte foot process architecture and loss of slit diaphragms while overall polarity is maintained. Nephrin, a major component of the slit diaphragm, is apically mis-localized in podocytes from crb2b morphants suggesting that crb2b is required for the proper protein trafficking of Nephrin. This report is the first to show a role for crb function in podocyte differentiation. Furthermore, these results suggest a novel link between epithelial polarization and the maintenance of a functional filtration barrier.

  14. Acute podocyte injury is not a stimulus for podocytes to migrate along the glomerular basement membrane in zebrafish larvae

    PubMed Central

    Siegerist, Florian; Blumenthal, Antje; Zhou, Weibin; Endlich, Karlhans; Endlich, Nicole

    2017-01-01

    Podocytes have a unique 3D structure of major and interdigitating foot processes which is the prerequisite for renal blood filtration. Loss of podocytes leads to chronic kidney disease ending in end stage renal disease. Until now, the question if podocytes can be replaced by immigration of cells along the glomerular basement membrane (GBM) is under debate. We recently showed that in contrast to former theories, podocytes are stationary in the zebrafish pronephros and neither migrate nor change their branching pattern of major processes over 23 hours. However, it was still unclear whether podocytes are able to migrate during acute injury. To investigate this, we applied the nitroreductase/metronidazole zebrafish model of podocyte injury to in vivo two-photon microscopy. The application of metronidazole led to retractions of major processes associated with a reduced expression of podocyte-specific proteins and a formation of subpodocyte pseudocyst. Electron microscopy showed that broad areas of the capillaries became denuded. By 4D in vivo observation of single podocytes, we could show that the remaining podocytes did not walk along GBM during 24 h. This in vivo study reveals that podocytes are very stationary cells making regenerative processes by podocyte walking along the GBM very unlikely. PMID:28252672

  15. Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species.

    PubMed

    Dahm, Ralf; Geisler, Robert

    2006-01-01

    In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.

  16. Dissection of the adult zebrafish kidney.

    PubMed

    Gerlach, Gary F; Schrader, Lauran N; Wingert, Rebecca A

    2011-08-29

    Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans. The zebrafish has become a powerful animal model for the study of vertebrate cell biology. There has been extensive documentation and analysis of embryonic development in the zebrafish. Only recently have scientists sought to document adult anatomy and surgical dissection techniques, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two

  17. Dissection of the Adult Zebrafish Kidney

    PubMed Central

    Wingert, Rebecca A.

    2011-01-01

    Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the

  18. Myomaker mediates fusion of fast myocytes in zebrafish embryos.

    PubMed

    Landemaine, Aurélie; Rescan, Pierre-Yves; Gabillard, Jean-Charles

    2014-09-05

    Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

  19. Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in zebrafish

    PubMed Central

    Cheng, Christina N.; Wingert, Rebecca A.

    2014-01-01

    The mechanisms that establish nephron segments are poorly understood. The zebrafish embryonic kidney, or pronephros, is a simplified yet conserved genetic model to study this renal development process because its nephrons contain segments akin to other vertebrates, including the proximal convoluted and straight tubules (PCT, PST). The zebrafish pronephros is also associated with the corpuscles of Stannius (CS), endocrine glands that regulate calcium and phosphate homeostasis, but whose ontogeny from renal progenitors is largely mysterious. Initial patterning of zebrafish renal progenitors in the intermediate mesoderm (IM) involves the formation of rostral and caudal domains, the former being reliant on retinoic acid (RA) signaling, and the latter being repressed by elevated RA levels. Here, using expression profiling to gain new insights into nephrogenesis, we discovered that the gene single minded family bHLH transcription factor 1a (sim1a) is dynamically expressed in the renal progenitors—first marking the caudal domain, then becoming restricted to the proximal segments, and finally exhibiting specific CS expression. In loss of function studies, sim1a knockdown expanded the PCT and abrogated both the PST and CS populations. Conversely, overexpression of sim1a modestly expanded the PST and CS, while it reduced the PCT. These results show that sim1a activity is necessary and partially sufficient to induce PST and CS fates, and suggest that sim1a may inhibit PCT fate and/or negotiate the PCT/PST boundary. Interestingly, the sim1a expression domain in renal progenitors is responsive to altered levels of RA, suggesting that RA regulates sim1a, directly or indirectly, during nephrogenesis. sim1a deficient embryos treated with exogenous RA formed nephrons that were predominantly composed of PCT segments, but lacked the enlarged PST observed in RA treated wild-types, indicating that RA is not sufficient to rescue the PST in the absence of sim1a expression. Alternately

  20. Nephron proximal tubule patterning and corpuscles of Stannius formation are regulated by the sim1a transcription factor and retinoic acid in zebrafish.

    PubMed

    Cheng, Christina N; Wingert, Rebecca A

    2015-03-01

    The mechanisms that establish nephron segments are poorly understood. The zebrafish embryonic kidney, or pronephros, is a simplified yet conserved genetic model to study this renal development process because its nephrons contain segments akin to other vertebrates, including the proximal convoluted and straight tubules (PCT, PST). The zebrafish pronephros is also associated with the corpuscles of Stannius (CS), endocrine glands that regulate calcium and phosphate homeostasis, but whose ontogeny from renal progenitors is largely mysterious. Initial patterning of zebrafish renal progenitors in the intermediate mesoderm (IM) involves the formation of rostral and caudal domains, the former being reliant on retinoic acid (RA) signaling, and the latter being repressed by elevated RA levels. Here, using expression profiling to gain new insights into nephrogenesis, we discovered that the gene single minded family bHLH transcription factor 1a (sim1a) is dynamically expressed in the renal progenitors-first marking the caudal domain, then becoming restricted to the proximal segments, and finally exhibiting specific CS expression. In loss of function studies, sim1a knockdown expanded the PCT and abrogated both the PST and CS populations. Conversely, overexpression of sim1a modestly expanded the PST and CS, while it reduced the PCT. These results show that sim1a activity is necessary and partially sufficient to induce PST and CS fates, and suggest that sim1a may inhibit PCT fate and/or negotiate the PCT/PST boundary. Interestingly, the sim1a expression domain in renal progenitors is responsive to altered levels of RA, suggesting that RA regulates sim1a, directly or indirectly, during nephrogenesis. sim1a deficient embryos treated with exogenous RA formed nephrons that were predominantly composed of PCT segments, but lacked the enlarged PST observed in RA treated wild-types, indicating that RA is not sufficient to rescue the PST in the absence of sim1a expression. Alternately

  1. Characterization of zebrafish dysferlin by morpholino knockdown

    SciTech Connect

    Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.; Alexander, Matthew S.; Kunkel, Louis M.

    2011-09-23

    Highlights: {yields} cDNAs of zebrafish dysferlin were cloned (6.3 kb). {yields} The dysferlin expression was detected in skeletal muscle, heart and eye. {yields} Injection of antisense morpholinos to dysferlin caused marked muscle disorganization. {yields} Zebrafish dysferlin expression may be involved in stabilizing muscle structures. -- Abstract: Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafish dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, {beta}-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.

  2. Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.

    PubMed

    Kalueff, Allan V; Gebhardt, Michael; Stewart, Adam Michael; Cachat, Jonathan M; Brimmer, Mallorie; Chawla, Jonathan S; Craddock, Cassandra; Kyzar, Evan J; Roth, Andrew; Landsman, Samuel; Gaikwad, Siddharth; Robinson, Kyle; Baatrup, Erik; Tierney, Keith; Shamchuk, Angela; Norton, William; Miller, Noam; Nicolson, Teresa; Braubach, Oliver; Gilman, Charles P; Pittman, Julian; Rosemberg, Denis B; Gerlai, Robert; Echevarria, David; Lamb, Elisabeth; Neuhauss, Stephan C F; Weng, Wei; Bally-Cuif, Laure; Schneider, Henning

    2013-03-01

    Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish 'do', and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.

  3. Towards a Comprehensive Catalog of Zebrafish Behavior 1.0 and Beyond

    PubMed Central

    Gebhardt, Michael; Stewart, Adam Michael; Cachat, Jonathan M.; Brimmer, Mallorie; Chawla, Jonathan S.; Craddock, Cassandra; Kyzar, Evan J.; Roth, Andrew; Landsman, Samuel; Gaikwad, Siddharth; Robinson, Kyle; Baatrup, Erik; Tierney, Keith; Shamchuk, Angela; Norton, William; Miller, Noam; Nicolson, Teresa; Braubach, Oliver; Gilman, Charles P.; Pittman, Julian; Rosemberg, Denis B.; Gerlai, Robert; Echevarria, David; Lamb, Elisabeth; Neuhauss, Stephan C.F.; Weng, Wei; Bally-Cuif, Laure; Schneider, Henning

    2013-01-01

    Abstract Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish ‘do’, and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species. PMID:23590400

  4. Study of Host–Microbe Interactions in Zebrafish

    PubMed Central

    Milligan-Myhre, Kathryn; Charette, Jeremy R.; Phennicie, Ryan T.; Stephens, W. Zac; Rawls, John F.; Guillemin, Karen; Kim, Carol H.

    2015-01-01

    All animals are ecosystems, home to diverse microbial populations. Animal-associated microbes play important roles in the normal development and physiology of their hosts, but can also be agents of infectious disease. Traditionally, mice have been used to study pathogenic and beneficial associations between microbes and vertebrate animals. The zebrafish is emerging as a valuable new model system for host-microbe interaction studies, affording researchers with the opportunity to survey large populations of hosts and to visualize microbe-host associations at a cellular level in living animals. This chapter provides detailed protocols for the analysis of zebrafish-associated microbial communities, the derivation and husbandry of germ-free zebrafish, and the modeling of infectious disease in different stages of zebrafish development via different routes of inoculation. These protocols offer a starting point for researchers to address a multitude of questions about animals’ coexistence with microorganisms. PMID:21951527

  5. Protocadherin-17 Function in Zebrafish Retinal Development

    PubMed Central

    Chen, Yun; Londraville, Richard; Brickner, Sarah; El-Shaar, Lana; Fankhauser, Kelsee; Dearth, Cassandra; Fulton, Leah; Sochacka, Alicja; Bhattarai, Sunil; Marrs, James A.; Liu, Qin

    2012-01-01

    Cadherin cell adhesion molecules play crucial roles in vertebrate development including the development of the retina. Most studies have focused on examining functions of classic cadherins (e.g. N-cadherin) in retinal development. There is little information on the function of protocadherins in the development of the vertebrate visual system. We previously showed that protocadherin-17 mRNA was expressed in developing zebrafish retina during critical stages of the retinal development. To gain insight into protocadherin-17 function in the formation of the retina, we analyzed eye development and differentiation of retinal cells in zebrafish embryos injected with protocadherin-17 specific antisense morpholino oligonucleotides (MOs). Protocadherin-17 knockdown embryos (pcdh17 morphants) had significantly reduced eyes due mainly to decreased cell proliferation. Differentiation of several retinal cell types (e.g. retinal ganglion cells) was also disrupted in the pcdh17 morphants. Phenotypic rescue was achieved by injection of protocadherin-17 mRNA. Injection of a vivo-protocadherin-17 MO into one eye of embryonic zebrafish resulted in similar eye defects. Our results suggest that protocadherin-17 plays an important role in the normal formation of the zebrafish retina. PMID:22927092

  6. Thyroid development in zebrafish lacking Taz.

    PubMed

    Pappalardo, Andrea; Porreca, Immacolata; Caputi, Luigi; De Felice, Elena; Schulte-Merker, Stephan; Zannini, Mariastella; Sordino, Paolo

    2015-11-01

    Taz is a signal-responsive transcriptional coregulator implicated in several biological functions, from chondrogenesis to regulation of organ size. Less well studied, however, is its role in thyroid formation. Here, we explored the in vivo effects on thyroid development of morpholino (MO)-mediated knockdown of wwtr1, the gene encoding zebrafish Taz. The wwtr1 gene is expressed in the thyroid primordium and pharyngeal tissue of developing zebrafish. Compared to mammalian cells, in which Taz promotes expression of thyroid transcription factors and thyroid differentiation genes, wwtr1 MO injection in zebrafish had little or no effect on the expression of thyroid transcription factors, and differentially altered the expression of thyroid differentiation genes. Analysis of wwtr1 morphants at later stages of development revealed that the number and the lumen of thyroid follicles, and the number of thyroid follicle cells, were significantly smaller. In addition, Taz-depleted larvae displayed patterning defects in ventral cranial vessels that correlate with lateral displacement of thyroid follicles. These findings indicate that the zebrafish Taz protein is needed for the normal differentiation of the thyroid and are the first to suggest that Taz confers growth advantage to the endocrine gland.

  7. Podocyte Developmental Defects Caused by Adriamycin in Zebrafish Embryos and Larvae: A Novel Model of Glomerular Damage

    PubMed Central

    Zennaro, Cristina; Mariotti, Massimo; Carraro, Michele; Pasqualetti, Sara; Corbelli, Alessandro; Armelloni, Silvia; Li, Min; Ikehata, Masami; Clai, Milan; Artero, Mary; Messa, Piergiorgio; Boscutti, Giuliano; Rastaldi, Maria Pia

    2014-01-01

    The zebrafish pronephros is gaining popularity in the nephrology community, because embryos are easy to cultivate in multiwell plates, allowing large number of experiments to be conducted in an in vivo model. In a few days, glomeruli reach complete development, with a structure that is similar to that of the mammalian counterpart, showing a fenestrated endothelium and a basement membrane covered by the multiple ramifications of mature podocytes. As a further advantage, zebrafish embryos are permeable to low molecular compounds, and this explains their extensive use in drug efficacy and toxicity experiments. Here we show that low concentrations of adriamycin (i.e. 10 and 20 µM), when dissolved in the medium of zebrafish embryos at 9 hours post-fertilization and removed after 48 hours (57 hpf), alter the development of podocytes with subsequent functional impairment, demonstrated by onset of pericardial edema and reduction of expression of the podocyte proteins nephrin and wt1. Podocyte damage is morphologically confirmed by electron microscopy and functionally supported by increased clearance of microinjected 70 kDa fluorescent dextran. Importantly, besides pericardial edema and glomerular damage, which persist and worsen after adriamycin removal from the medium, larvae exposed to adriamycin 10 and 20 µM do not show any myocardiocyte alterations nor vascular changes. The only extra-renal effect is a transient delay of cartilage formation that rapidly recovers once adriamycin is removed. In summary, this low dose adriamycin model can be applied to analyze podocyte developmental defects, such as those observed in congenital nephrotic syndrome, and can be taken in consideration for pharmacological studies of severe early podocyte injury. PMID:24845233

  8. Measuring zebrafish turning rate.

    PubMed

    Mwaffo, Violet; Butail, Sachit; di Bernardo, Mario; Porfiri, Maurizio

    2015-06-01

    Zebrafish is becoming a popular animal model in preclinical research, and zebrafish turning rate has been proposed for the analysis of activity in several domains. The turning rate is often estimated from the trajectory of the fish centroid that is output by commercial or custom-made target tracking software run on overhead videos of fish swimming. However, the accuracy of such indirect methods with respect to the turning rate associated with changes in heading during zebrafish locomotion is largely untested. Here, we compare two indirect methods for the turning rate estimation using the centroid velocity or position data, with full shape tracking for three different video sampling rates. We use tracking data from the overhead video recorded at 60, 30, and 15 frames per second of zebrafish swimming in a shallow water tank. Statistical comparisons of absolute turning rate across methods and sampling rates indicate that, while indirect methods are indistinguishable from full shape tracking, the video sampling rate significantly influences the turning rate measurement. The results of this study can aid in the selection of the video capture frame rate, an experimental design parameter in zebrafish behavioral experiments where activity is an important measure.

  9. Dystrophic muscle improvement in zebrafish via increased heme oxygenase signaling

    PubMed Central

    Kawahara, Genri; Gasperini, Molly J.; Myers, Jennifer A.; Widrick, Jeffrey J.; Eran, Alal; Serafini, Peter R.; Alexander, Matthew S.; Pletcher, Mathew T.; Morris, Carl A.; Kunkel, Louis M.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx5cv mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications. PMID:24234649

  10. Heart-specific expression of laminopathic mutations in transgenic zebrafish.

    PubMed

    Verma, Ajay D; Parnaik, Veena K

    2017-07-01

    Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

  11. Feed and Feeding Regime Affect Growth Rate and Gonadosomatic Index of Adult Zebrafish (Danio Rerio)

    PubMed Central

    Law, Sheran Hiu Wan

    2013-01-01

    Abstract A 5-week study was conducted to evaluate commercially available Artemia, Ziegler zebrafish diet, and Calamac diet fed in five different feeding regimes on the growth and reproductive development of 7-month-old zebrafish. Zebrafish were fed to satiation three times daily during the normal work week and twice daily during the weekend and holidays. Zebrafish in dietary groups CCC (Calamac three times daily) and CCA (Calamac twice daily, Artemia once daily) had a significantly (p<0.05) greater weight gain and specific growth rate as compared to all other dietary groups. Male zebrafish in dietary group 5 had significantly larger gonadosomatic index (GSI) values than all other groups, while female zebrafish in dietary group CCC had significantly larger GSI values than all other groups. No differences in the fatty acid content of female gonads were detected. Zebrafish fed solely Artemia had the greatest weight loss and lowest GSI values. Preliminary evidence of protein sparing in zebrafish is reported. Collectively, this study sheds more light into the effects of the use of commercially available feeds and feeding regime on the rearing of zebrafish. PMID:23902461

  12. Feed and feeding regime affect growth rate and gonadosomatic index of adult zebrafish (Danio rerio).

    PubMed

    Gonzales, John M; Law, Sheran Hiu Wan

    2013-12-01

    A 5-week study was conducted to evaluate commercially available Artemia, Ziegler zebrafish diet, and Calamac diet fed in five different feeding regimes on the growth and reproductive development of 7-month-old zebrafish. Zebrafish were fed to satiation three times daily during the normal work week and twice daily during the weekend and holidays. Zebrafish in dietary groups CCC (Calamac three times daily) and CCA (Calamac twice daily, Artemia once daily) had a significantly (p<0.05) greater weight gain and specific growth rate as compared to all other dietary groups. Male zebrafish in dietary group 5 had significantly larger gonadosomatic index (GSI) values than all other groups, while female zebrafish in dietary group CCC had significantly larger GSI values than all other groups. No differences in the fatty acid content of female gonads were detected. Zebrafish fed solely Artemia had the greatest weight loss and lowest GSI values. Preliminary evidence of protein sparing in zebrafish is reported. Collectively, this study sheds more light into the effects of the use of commercially available feeds and feeding regime on the rearing of zebrafish.

  13. Mycobacteriosis in zebrafish colonies.

    PubMed

    Whipps, Christopher M; Lieggi, Christine; Wagner, Robert

    2012-01-01

    Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward.

  14. Mycobacteriosis in Zebrafish Colonies

    PubMed Central

    Whipps, Christopher M.; Lieggi, Christine; Wagner, Robert

    2016-01-01

    Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward. PMID:23382341

  15. [Establishment of a diet-induced obesity model in zebrafish larvae].

    PubMed

    Zheng, Xinchun; Liu, Li; Dai, Wencong; Wang, Kunyuan; Chen, Xiaohui; Zhao, Lingfeng; Huang, Zhibin; Hou, Jinlin

    2016-01-01

    To establish a diet-induced obesity model in zebrafish larvae. At 7 days post-fertilization (dpf), 200 zebrafish larvae with normal development were randomly allocated to two groups with the feeding quantity of 30 mg per day (normal feeding group) or 180 mg per day (overfed group) for 20 days. The weight, length, BMI, triglyceride (TG) and total cholesterol (TCH) of each group were measured. Whole-mount Oil Red O staining, frozen Oil Red O staining and hematoxylin-eosin (HE) staining were used to estimate the rate of hepatic steatosis and liver histology of the zebrafish. The dynamic change of hepatic lipid droplets and distribution of adipose tissue were observed with Nile Red staining in overfed zebrafish in vivo. The weight, length, BMI and TG of overfed zebrafish were significantly increased compared with those in normal feeding group. Whole-mount Oil Red O staining showed that the percent of hepatic steatosis in overfed group (89.4%) was markedly higher than that in normal feeding group (20.7%). Macrovesicular steatosis was observed in the liver of the overfed larvae. Nile Red staining visualized hepatic lipid droplets and the distribution of larval adipose tissue, which increased with feeding time in the overfed zebrafish. Starving larvae showed depletion of fat and hepatic lipid, and adipose tissue was induced after refeeding. We successfully established an diet-induced obesity model in zebrafish larva, in which Nile Red staining allows in vivo observation of the adipocytes and hepatic lipid droplets.

  16. Myomaker mediates fusion of fast myocytes in zebrafish embryos

    SciTech Connect

    Landemaine, Aurélie; Rescan, Pierre-Yves; Gabillard, Jean-Charles

    2014-09-05

    Highlights: • Myomaker is transiently expressed in fast myocytes during embryonic myogenesis. • Myomaker is essential for fast myocyte fusion in zebrafish. • The function of myomaker is conserved among Teleostomi. - Abstract: Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

  17. T cell immune deficiency in zap70 mutant zebrafish.

    PubMed

    Moore, John C; Mulligan, Timothy S; Torres Yordán, Nora; Castranova, Daniel; Pham, Van N; Tang, Qin; Lobbardi, Riadh; Anselmo, Anthony; Liwski, Robert S; Berman, Jason N; Sadreyev, Ruslan I; Weinstein, Brant M; Langenau, David M

    2016-09-06

    The zeta-chain (TCR) associated protein kinase 70kDa (ZAP70) is required for T-cell activation. ZAP70 deficiencies in humans and null mutations in mice lead to severe combined immunodeficiency. Here, we describe a zap70 loss-of-function mutation in zebrafish (zap70(y442)) that was created using TALENs. In contrast to what has been reported in morphant zebrafish, zap70(y442) homozygous mutant zebrafish displayed normal development of blood and lymphatic vasculature. Hematopoietic cell development was also largely unaffected in mutant larvae. However, mutant fish had reduced lck:GFP+ thymic T cells by 5 days post-fertilization that persisted into adult stages. Morphological analysis, RNA sequencing and single-cell gene expression profiling of whole kidney marrow cells of adult fish revealed complete loss of mature T cells in zap70(y442) mutant animals. T cell immunodeficiency was confirmed through transplantation of unmatched normal and malignant donor cells into zap70(y442) mutant zebrafish, with T cell loss being sufficient for robust allogeneic cell engraftment. zap70 mutant zebrafish show remarkable conservation of immune cell dysfunction as found in mice and humans and will serve as a valuable model to study zap70 immunodeficiency. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  18. Somitogenesis in zebrafish.

    PubMed

    Holley, S A; Nüsslein-Volhard, C

    2000-01-01

    Both genetic and embryological studies in the zebrafish, Danio rerio, have contributed to our general understanding of how somites form and differentiate. In the zebrafish, mutants have been isolated that have specific effects on virtually every aspect of somite development. The fss-type mutants, defining 5 genes, affect somite segmentation and epithelialization. The you-type mutants, comprising 7 genes, and mutants in another 13 genes defective in notochord formation, have somites with abnormal pattern and morphology. Eighteen genes have been identified that are required for the differentiation and maintenance of the somitic musculature, and 2 genes have been identified that are involved in the development of motoneurons that innervate the somitic musculature. The true utility of the zebrafish lies in the ability to combine genetic analysis with embryological experimentation. Such analysis of somite segmentation suggests that homologues of both the Drosophila pair-rule and segment polarity genes, her1 and Sonic hedge-hog, respectively, are involved generating periodicity during somitogenesis. The Sonic hedge-hog protein secreted from the notochord also induces the formation of specific muscle types including the slow muscle fibers which are initially induced in the medial somite and undergo a series of morphological transitions including migration through the somite to the lateral surface where they complete their differentiation. The role of the notochord in patterning the somite is also demonstrated by its involvement in regulating the permissiveness of the somite to the extension of axons of primary motoneurons.

  19. A Retrospective Study of the Prevalence and Classification of Intestinal Neoplasia in Zebrafish (Danio Rerio)

    PubMed Central

    Paquette, Colleen E.; Buchner, Cari; Tanguay, Robert L.; Guillemin, Karen; Mason, Timothy J.; Peterson, Tracy S.

    2013-01-01

    Abstract For over a decade, spontaneous intestinal neoplasia has been observed in zebrafish (Danio rerio) submitted to the ZIRC (Zebrafish International Resource Center) diagnostic service. In addition, zebrafish displayed preneoplastic intestinal changes including hyperplasia, dysplasia, and enteritis. A total of 195 zebrafish, representing 2% of the total fish submitted to the service, were diagnosed with these lesions. Neoplastic changes were classified either as adenocarcinoma or small cell carcinoma, with a few exceptions (carcinoma not otherwise specified, tubular adenoma, and tubulovillous adenoma). Tumor prevalence appeared similarly distributed between sexes and generally occurred in zebrafish greater than 1 year of age, although neoplastic changes were observed in fish 6 months of age. Eleven lines displayed these preneoplastic and neoplastic changes, including wild-types and mutants. Affected zebrafish originated from 18 facilities, but the majority of fish were from a single zebrafish research facility (hereafter referred to as the primary facility) that has submitted numerous samples to the ZIRC diagnostic service. Zebrafish from the primary facility submitted as normal sentinel fish demonstrate that these lesions are most often subclinical. Fish fed the diet from the primary facility and held at another location did not develop intestinal lesions, indicating that diet is not the etiologic agent. PMID:23544991

  20. A retrospective study of the prevalence and classification of intestinal neoplasia in zebrafish (Danio rerio).

    PubMed

    Paquette, Colleen E; Kent, Michael L; Buchner, Cari; Tanguay, Robert L; Guillemin, Karen; Mason, Timothy J; Peterson, Tracy S

    2013-06-01

    For over a decade, spontaneous intestinal neoplasia has been observed in zebrafish (Danio rerio) submitted to the ZIRC (Zebrafish International Resource Center) diagnostic service. In addition, zebrafish displayed preneoplastic intestinal changes including hyperplasia, dysplasia, and enteritis. A total of 195 zebrafish, representing 2% of the total fish submitted to the service, were diagnosed with these lesions. Neoplastic changes were classified either as adenocarcinoma or small cell carcinoma, with a few exceptions (carcinoma not otherwise specified, tubular adenoma, and tubulovillous adenoma). Tumor prevalence appeared similarly distributed between sexes and generally occurred in zebrafish greater than 1 year of age, although neoplastic changes were observed in fish 6 months of age. Eleven lines displayed these preneoplastic and neoplastic changes, including wild-types and mutants. Affected zebrafish originated from 18 facilities, but the majority of fish were from a single zebrafish research facility (hereafter referred to as the primary facility) that has submitted numerous samples to the ZIRC diagnostic service. Zebrafish from the primary facility submitted as normal sentinel fish demonstrate that these lesions are most often subclinical. Fish fed the diet from the primary facility and held at another location did not develop intestinal lesions, indicating that diet is not the etiologic agent.

  1. Acute restraint stress in zebrafish: behavioral parameters and purinergic signaling.

    PubMed

    Piato, Angelo L; Rosemberg, Denis B; Capiotti, Katiucia M; Siebel, Anna M; Herrmann, Ana P; Ghisleni, Gabriele; Vianna, Monica R; Bogo, Maurício R; Lara, Diogo R; Bonan, Carla D

    2011-10-01

    Despite the extensive knowledge about the effects of acute restraint stress (ARS) in rodents, zebrafish research is still elementary in this field, and the consequences of stress on purinergic system are unclear. Therefore, we evaluated the effects of ARS on behavior, biochemical, and molecular parameters in zebrafish brain. Animals were submitted to a 90 min ARS protocol and tested for anxiety levels, exploratory behavior, and memory performance. Furthermore, we analyzed ectonucleotidase and adenosine deaminase activities and their gene expression profile, as well as transcription of adenosine receptors. ARS increased anxiety, but did not impair locomotion or cognition. ARS significantly increased ATP hydrolysis, decreased cytosolic ADA activity, and changed the entpd and adora gene expression. In conclusion, ARS disturbed zebrafish behavior, and we hypothesize that the augmentation in adenosine-mediated signaling may be a strategy to reestablish homeostasis and normal behavior after a stressful event.

  2. Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

    PubMed Central

    Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

    2017-01-01

    Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

  3. Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

    PubMed

    Pylatiuk, Christian; Sanchez, Daniela; Mikut, Ralf; Alshut, Rüdiger; Reischl, Markus; Hirth, Sofia; Rottbauer, Wolfgang; Just, Steffen

    2014-08-01

    A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.

  4. Nicotinic involvement in memory function in zebrafish.

    PubMed

    Levin, Edward D; Chen, Elaine

    2004-01-01

    Zebrafish are an emerging model for the study of the molecular mechanisms of brain function. To conduct studies of the neural bases of behavior in zebrafish, we must understand the behavioral function of zebrafish and how it is altered by perturbations of brain function. This study determined nicotine actions on memory function in zebrafish. With the methods that we have developed to assess memory in zebrafish using delayed spatial alternation (DSA), we determined the dose effect function of acute nicotine on memory function in zebrafish. As in rodents and primates, low nicotine doses improve memory in zebrafish, while high nicotine doses have diminished effect and can impair memory. This study shows that nicotine affects memory function in zebrafish much like in rats, mice, monkeys and humans. Now, zebrafish can be used to help understand the molecular mechanisms crucial to nicotine effects on memory.

  5. GROWTH AND BEHAVIOR OF LARVAL ZEBRAFISH Danio ...

    EPA Pesticide Factsheets

    Because Zebrafish (Danio rerio) have become a popular and important model for scientific research, the capability to rear larval zebrafish to adulthood is of great importance. Recently research examining the effects of diet (live versus processed) have been published. In the current study we examined whether the larvae can be reared on a processed diet alone, live food alone, or the combination while maintaining normal locomotor behavior, and acceptable survival, length and weight at 14 dpf in a static system. A 14 day feeding trial was conducted in glass crystallizing dishes containing 500 ml of 4 ppt Instant Ocean. On day 0 pdf 450 embryos were selected as potential study subjects and placed in a 26○C incubator on a 14:10 (light:dark) light cycle. At 4 dpf 120 normally developing embryos were selected per treatment and divided into 3 bowls of 40 embryos (for an n=3 per treatment; 9 bowls total). Treatment groups were: G (Gemma Micro 75 only), R (L-type marine rotifers (Brachionus plicatilis) only) or B (Gemma and rotifers). Growth (length), survival, water quality and rotifer density were monitored on days 5-14. On day 14, weight of larva in each bowl was measured and 8 larva per bowl were selected for use in locomotor testing. This behavior paradigm tests individual larval zebrafish under both light and dark conditions in a 24-well plate.After 14 dpf, survival among the groups was not different (92-98%). By days 7 -14 R and B larvae were ~2X longer

  6. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  7. Fish-on-a-chip: microfluidics for zebrafish research.

    PubMed

    Yang, Fan; Gao, Chuan; Wang, Ping; Zhang, Guo-Jun; Chen, Zuanguang

    2016-04-07

    High-efficiency zebrafish (embryo) handling platforms are crucially needed to facilitate the deciphering of the increasingly expanding vertebrate-organism model values. However, the manipulation platforms for zebrafish are scarce and rely mainly on the conventional "static" microtiter plates or glass slides with rigid gel, which limits the dynamic, three-dimensional (3D), tissue/organ-oriented information acquisition from the intact larva with normal developmental dynamics. In addition, these routine platforms are not amenable to high-throughput handling of such swimming multicellular biological entities at the single-organism level and incapable of precisely controlling the growth microenvironment by delivering stimuli in a well-defined spatiotemporal fashion. Recently, microfluidics has been developed to address these technical challenges via tailor-engineered microscale structures or structured arrays, which integrate with or interface to functional components (e.g. imaging systems), allowing quantitative readouts of small objects (zebrafish larvae and embryos) under normal physiological conditions. Here, we critically review the recent progress on zebrafish manipulation, imaging and phenotype readouts of external stimuli using these microfluidic tools and discuss the challenges that confront these promising "fish-on-a-chip" technologies. We also provide an outlook on future potential trends in this field by combining with bionanoprobes and biosensors.

  8. The Effect of Zeaxanthin on the Visual Acuity of Zebrafish.

    PubMed

    Saidi, Eric A; Davey, Pinakin Gunvant; Cameron, D Joshua

    2015-01-01

    Oral supplementation of carotenoids such as zeaxanthin or lutein which naturally occur in human retina have been shown to improve vision and prevent progression of damage to advanced AMD in some studies. The zebrafish eye shares many physiological similarities with the human eye and is increasingly being used as model for vision research. We hypothesized that injection of zeaxanthin into the zebrafish eye would improve the visual acuity of the zebrafish over time. Visual acuity, calculated in cycles per degree, was measured in adult zebrafish to establish a consistent baseline using the optokinetic response. Zeaxanthin dissolved into phosphate buffered saline (PBS) or PBS only was injected into the anterior chamber of the right and left eyes of the Zebrafish. Visual acuities were measured at 1 week and 3, 8 and 12 weeks post-injection to compare to baseline values. Repeated measures ANOVA was used to compare visual acuities between fish injected with PBS and zeaxanthin. A significant improvement in visual acuity, 14% better than before the injection (baseline levels), was observed one week after injection with zeaxanthin (p = 0.04). This improvement peaked at more than 30% for some fish a few weeks after the injection and improvement in vision persisted at 3 weeks after injection (p = 0.006). The enhanced visual function was not significantly better than baseline at 8 weeks (p = 0.19) and returned to baseline levels 12 weeks after the initial injection (p = 0.50). Zeaxanthin can improve visual acuity in zebrafish eyes. Further studies are required to develop a better understanding of the role zeaxanthin and other carotenoids play during normal visual function.

  9. Persistent Behavioral Impairment Caused by Embryonic Methylphenidate Exposure in Zebrafish

    PubMed Central

    Levin, Edward D.; Sledge, Damiyon; Roach, Stephanie; Petro, Ann; Donerly, Susan; Linney, Elwood

    2011-01-01

    As more adults take the stimulant medication methylphenidate to treat attention deficit hyperactivity disorder (ADHD) residual type, the risk arises with regard to the potential risks of early developmental exposure if people taking the medication become pregnant. We studied the neurobehavioral effects of methylphenidate in zebrafish. Zebrafish offer cellular reporter systems, continuous visual access and molecular interventions such as morpholinos to help determine critical mechanisms underlying neurobehavioral teratogenicity. Previously, we had seen that persisting neurobehavioral impairment in zebrafish with developmental chlorpyrifos exposure was associated with disturbed dopamine systems. Because methylphenidate is an indirect dopamine agonist, it was thought that it might also cause persistent behavioral impairment after developmental exposure. Zebrafish embryos were exposed to the ADHD stimulant medication methylphenidate 0-5 days post fertilization (12.5-50 mg/l). They were tested for long-term behavioral effects as adults. Methylphenidate exposure (50 mg/l) caused significant increases in dopamine, norepinepherine and serotonin on day 6 but not day 30 after fertilization. In the novel tank diving test of predatory avoidance developmental methylphenidate (50 mg/l) caused a significant reduction in the normal diving response. In the three-chamber spatial learning task early developmental methylphenidate (50 mg/l) caused a significant impairment in choice accuracy. These data show that early developmental exposure of zebrafish to methylphenidate causes a long-term impairment in neurobehavioral plasticity. The identification of these functional deficits in zebrafish enables further studies with this model to determine how molecular and cellular mechanisms are disturbed to arrive at this compromised state. PMID:21741476

  10. Genetic Analysis of Histamine Signaling in Larval Zebrafish Sleep

    PubMed Central

    Oikonomou, Grigorios

    2017-01-01

    Abstract Pharmacological studies in mammals and zebrafish suggest that histamine plays an important role in promoting arousal. However, genetic studies using rodents with disrupted histamine synthesis or signaling have revealed only subtle or no sleep/wake phenotypes. Studies of histamine function in mammalian arousal are complicated by its production in cells of the immune system and its roles in humoral and cellular immunity, which can have profound effects on sleep/wake states. To avoid this potential confound, we used genetics to explore the role of histamine in regulating sleep in zebrafish, a diurnal vertebrate in which histamine production is restricted to neurons in the brain. Similar to rodent genetic studies, we found that zebrafish that lack histamine due to mutation of histidine decarboxylase (hdc) exhibit largely normal sleep/wake behaviors. Zebrafish containing predicted null mutations in several histamine receptors also lack robust sleep/wake phenotypes, although we are unable to verify that these mutants are completely nonfunctional. Consistent with some rodent studies, we found that arousal induced by overexpression of the neuropeptide hypocretin (Hcrt) or by stimulation of hcrt-expressing neurons is not blocked in hdc or hrh1 mutants. We also found that the number of hcrt-expressing or histaminergic neurons is unaffected in animals that lack histamine or Hcrt signaling, respectively. Thus, while acute pharmacological manipulation of histamine signaling has been shown to have profound effects on zebrafish and mammalian sleep, our results suggest that chronic loss of histamine signaling due to genetic mutations has only subtle effects on sleep in zebrafish, similar to rodents. PMID:28275716

  11. Persistent behavioral impairment caused by embryonic methylphenidate exposure in zebrafish.

    PubMed

    Levin, Edward D; Sledge, Damiyon; Roach, Stephanie; Petro, Ann; Donerly, Susan; Linney, Elwood

    2011-01-01

    As more adults take the stimulant medication methylphenidate to treat attention deficit hyperactivity disorder (ADHD) residual type, the risk arises with regard to exposure during early development if people taking the medication become pregnant. We studied the neurobehavioral effects of methylphenidate in zebrafish. Zebrafish offer cellular reporter systems, continuous visual access and molecular interventions such as morpholinos to help determine critical mechanisms underlying neurobehavioral teratogenicity. Previously, we had seen that persisting neurobehavioral impairment in zebrafish with developmental chlorpyrifos exposure was associated with disturbed dopamine systems. Because methylphenidate is an indirect dopamine agonist, it was thought that it might also cause persistent behavioral impairment after developmental exposure. Zebrafish embryos were exposed to the ADHD stimulant medication methylphenidate 0-5 days post fertilization (12.5-50mg/l). They were tested for long-term behavioral effects as adults. Methylphenidate exposure (50mg/l) caused significant increases in dopamine, norepinepherine and serotonin on day 6 but not day 30 after fertilization. In the novel tank diving test of predatory avoidance developmental methylphenidate (50mg/l) caused a significant reduction in the normal diving response. In the three-chamber spatial learning task early developmental methylphenidate (50mg/l) caused a significant impairment in choice accuracy. These data show that early developmental exposure of zebrafish to methylphenidate causes a long-term impairment in neurobehavioral plasticity. The identification of these functional deficits in zebrafish enables further studies with this model to determine how molecular and cellular mechanisms are disturbed to arrive at this compromised state.

  12. Protective effect of rutin on impairment of cognitive functions of due to antiepileptic drugs on zebrafish model.

    PubMed

    Dubey, Shagun; Ganeshpurkar, Aditya; Bansal, Divya; Dubey, Nazneen

    2015-01-01

    The severity of adverse reactions due to antiepileptics is observed during initiation and early treatment in which impairment of cognitive effects are common. Since long time, herbal medicine is a natural remedy to treat neural symptoms. Phytochemicals have been proven to be potent neuro-protective agents. Rutin, a bioflavonoid is established to be nootropic in many studies. In this study, we aimed to determine the protective effect of rutin in zebrafish against the side effects produced by AEDs. Seizures were induced in zebrafish by phenylenetetrazole. Rutin pretreatment (50 mg/kg, single injection, i.p.) was done before commencement of the study. Behavioral studies were performed as: latency to move high in the tank, locomotion effects, color effect, shoal cohesion, light/dark test on Zebrafish. Treatment with rutin reverted the locomotor behavior to normal. Treatment with AEDs caused fishes to move in all regions while, in case of treatment with rutin, the response reverted to normal. Treatment with AEDs altered swimming behavior of zebrafish, however, rutin showed a positive effect over this behavior. Treatment with AEDs resulted in restricted movement of zebrafish to the dark zone. Treatment with rutin caused increased latency of zebrafish to move in the light compartment. Similarly, time spent in the light compartment by zebrafish treated with rutin was significantly (P < 0.01) higher as compared to zebrafish treated with AEDs. The results suggest a protective role of rutin on cognition impaired by AEDs.

  13. Thrombin Generation in Zebrafish Blood

    PubMed Central

    Hemker, Coenraad; Lindhout, Theo; Kelchtermans, Hilde; de Laat, Bas

    2016-01-01

    To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis. PMID:26872266

  14. Identification and Expression Analysis of the Complete Family of Zebrafish pkd Genes.

    PubMed

    England, Samantha J; Campbell, Paul C; Banerjee, Santanu; Swanson, Annika J; Lewis, Katharine E

    2017-01-01

    Polycystic kidney disease (PKD) proteins are trans-membrane proteins that have crucial roles in many aspects of vertebrate development and physiology, including the development of many organs as well as left-right patterning and taste. They can be divided into structurally-distinct PKD1-like and PKD2-like proteins and usually one PKD1-like protein forms a heteromeric polycystin complex with a PKD2-like protein. For example, PKD1 forms a complex with PKD2 and mutations in either of these proteins cause Autosomal Dominant Polycystic Kidney Disease (ADPKD), which is the most frequent potentially-lethal single-gene disorder in humans. Here, we identify the complete family of pkd genes in zebrafish and other teleosts. We describe the genomic locations and sequences of all seven genes: pkd1, pkd1b, pkd1l1, pkd1l2a, pkd1l2b, pkd2, and pkd2l1. pkd1l2a/pkd1l2b are likely to be ohnologs of pkd1l2, preserved from the whole genome duplication that occurred at the base of the teleosts. However, in contrast to mammals and cartilaginous and holostei fish, teleosts lack pkd2l2, and pkdrej genes, suggesting that these have been lost in the teleost lineage. In addition, teleost, and holostei fish have only a partial pkd1l3 sequence, suggesting that this gene may be in the process of being lost in the ray-finned fish lineage. We also provide the first comprehensive description of the expression of zebrafish pkd genes during development. In most structures we detect expression of one pkd1-like gene and one pkd2-like gene, consistent with these genes encoding a heteromeric protein complex. For example, we found that pkd2 and pkd1l1 are expressed in Kupffer's vesicle and pkd1 and pkd2 are expressed in the developing pronephros. In the spinal cord, we show that pkd1l2a and pkd2l1 are co-expressed in KA cells. We also identify potential co-expression of pkd1b and pkd2 in the floor-plate. Interestingly, and in contrast to mouse, we observe expression of all seven pkd genes in regions that

  15. Identification and Expression Analysis of the Complete Family of Zebrafish pkd Genes

    PubMed Central

    England, Samantha J.; Campbell, Paul C.; Banerjee, Santanu; Swanson, Annika J.; Lewis, Katharine E.

    2017-01-01

    Polycystic kidney disease (PKD) proteins are trans-membrane proteins that have crucial roles in many aspects of vertebrate development and physiology, including the development of many organs as well as left–right patterning and taste. They can be divided into structurally-distinct PKD1-like and PKD2-like proteins and usually one PKD1-like protein forms a heteromeric polycystin complex with a PKD2-like protein. For example, PKD1 forms a complex with PKD2 and mutations in either of these proteins cause Autosomal Dominant Polycystic Kidney Disease (ADPKD), which is the most frequent potentially-lethal single-gene disorder in humans. Here, we identify the complete family of pkd genes in zebrafish and other teleosts. We describe the genomic locations and sequences of all seven genes: pkd1, pkd1b, pkd1l1, pkd1l2a, pkd1l2b, pkd2, and pkd2l1. pkd1l2a/pkd1l2b are likely to be ohnologs of pkd1l2, preserved from the whole genome duplication that occurred at the base of the teleosts. However, in contrast to mammals and cartilaginous and holostei fish, teleosts lack pkd2l2, and pkdrej genes, suggesting that these have been lost in the teleost lineage. In addition, teleost, and holostei fish have only a partial pkd1l3 sequence, suggesting that this gene may be in the process of being lost in the ray-finned fish lineage. We also provide the first comprehensive description of the expression of zebrafish pkd genes during development. In most structures we detect expression of one pkd1-like gene and one pkd2-like gene, consistent with these genes encoding a heteromeric protein complex. For example, we found that pkd2 and pkd1l1 are expressed in Kupffer's vesicle and pkd1 and pkd2 are expressed in the developing pronephros. In the spinal cord, we show that pkd1l2a and pkd2l1 are co-expressed in KA cells. We also identify potential co-expression of pkd1b and pkd2 in the floor-plate. Interestingly, and in contrast to mouse, we observe expression of all seven pkd genes in regions

  16. Knockdown of Zebrafish Blood Vessel Epicardial Substance Results in Incomplete Retinal Lamination

    PubMed Central

    Chen, Ruei-Feng; Liu, Chia-Yang; Hu, Fung Rong; Huang, Chang-Jen; Wang, I-Jong

    2014-01-01

    Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. In vertebrates, blood vessel epicardial substance (Bves) is known to play an important role in the formation and maintenance of the tight junctions essential for epithelial cell polarity. In the current study, we generated a transgenic zebrafish Bves (zbves) promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinal lamination. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were used to identify the morphological defects in normal and Bves knockdown zebrafish. In normal zebrafish, Bves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer (IPL) and retinal pigment epithelium (RPE). In contrast, a loss of normal retinal lamination and cellular polarity was found with undifferentiated photoreceptor cells in Bves knockdown zebrafish. Herein, our results indicated that disruption of Bves will result in a loss of normal retinal lamination. PMID:24741362

  17. Zebrafish regenerate full thickness optic nerve myelin after demyelination, but this fails with increasing age.

    PubMed

    Münzel, Eva Jolanda; Becker, Catherina G; Becker, Thomas; Williams, Anna

    2014-07-15

    In the human demyelinating central nervous system (CNS) disease multiple sclerosis, remyelination promotes recovery and limits neurodegeneration, but this is inefficient and always ultimately fails. Furthermore, these regenerated myelin sheaths are thinner and shorter than the original, leaving the underlying axons potentially vulnerable. In rodent models, CNS remyelination is more efficient, so that in young animals (but not old) the number of myelinated axons is efficiently restored to normal, but in both young and old rodents, regenerated myelin sheaths are still short and thin. The reasons for these differences in remyelination efficiency, the thinner remyelinated myelin sheaths compared to developmental myelin and the subsequent effect on the underlying axon are unclear. We studied CNS remyelination in the highly regenerative adult zebrafish (Danio rerio), to better understand mechanisms of what we hypothesised would be highly efficient remyelination, and to identify differences to mammalian CNS remyelination, as larval zebrafish are increasingly used for high throughput screens to identify potential drug targets to improve myelination and remyelination. We developed a novel method to induce a focal demyelinating lesion in adult zebrafish optic nerve with no discernible axonal damage, and describe the cellular changes over time. Remyelination is indeed efficient in both young and old adult zebrafish optic nerves, and at 4 weeks after demyelination, the number of myelinated axons is restored to normal, but internode lengths are short. However, unlike in rodents or in humans, in young zebrafish these regenerated myelin sheaths were of normal thickness, whereas in aged zebrafish, they were thin, and remained so even 3 months later. This inability to restore normal myelin thickness in remyelination with age was associated with a reduced macrophage/microglial response. Zebrafish are able to efficiently restore normal thickness myelin around optic nerve axons after

  18. Dynamic glucoregulation and mammalian-like responses to metabolic and developmental disruption in zebrafish

    PubMed Central

    Jurczyk, Agata; Roy, Nicole; Bajwa, Rabia; Gut, Philipp; Lipson, Kathryn; Yang, Chaoxing; Covassin, Laurence; Racki, Waldemar J.; Rossini, Aldo A.; Phillips, Nancy; Stainier, Didier Y. R.; Greiner, Dale L.; Brehm, Michael A.; Bortell, Rita; diIorio, Philip

    2010-01-01

    Zebrafish embryos are emerging as models of glucose metabolism. However, patterns of endogenous glucose levels, and the role of the islet in glucoregulation, are unknown. We measured absolute glucose levels in zebrafish and mouse embryos, and demonstrate similar, dynamic glucose fluctuations in both species. Further, we show that chemical and genetic perturbations elicit mammalian-like glycemic responses in zebrafish embryos. We show that glucose is undetectable in early zebrafish and mouse embryos, but increases in parallel with pancreatic islet formation in both species. In zebrafish, increasing glucose is associated with activation of gluconeogenic phosphoenolpyruvate carboxykinase1 (pck1) transcription. Non-hepatic Pck1 protein is expressed in mouse embryos. We show, using RNA in situ hybridization, that zebrafish pck1 mRNA is similarly expressed in multiple cell types prior to hepatogenesis. Further, we demonstrate that the Pck1 inhibitor 3-mercaptopicolinic acid suppresses normal glucose accumulation in early zebrafish embryos. This shows that pre- and extra-hepatic pck1 is functional, and provides glucose locally to rapidly developing tissues. To determine if the primary islet is glucoregulatory in early fish embryos, we injected pdx1-specific morpholinos into transgenic embryos expressing GFP in beta cells. Most morphant islets were hypomorphic, not agenetic, but embryos still exhibited persistent hyperglycemia. We conclude from these data that the early zebrafish islet is functional, and regulates endogenous glucose. In summary, we identify mechanisms of glucoregulation in zebrafish embryos that are conserved with embryonic and adult mammals. These observations justify use of this model in mechanistic studies of human metabolic disease. PMID:20965191

  19. Zebrafish (Danio rerio) bioassay for visceral toxicosis of catfish and botulinum neurotoxin serotype E.

    PubMed

    Chatla, Kamalakar; Gaunt, Patricia; Petrie-Hanson, Lora; Hohn, Claudia; Ford, Lorelei; Hanson, Larry

    2014-03-01

    Visceral toxicosis of catfish (VTC), a sporadic disease of cultured channel catfish (Ictalurus punctatus) often with high mortality, is caused by botulinum neurotoxin serotype E (BoNT/E). Presumptive diagnosis of VTC is based on characteristic clinical signs and lesions, and the production of these signs and mortality after sera from affected fish is administered to sentinel catfish. The diagnosis is confirmed if the toxicity is neutralized with BoNT/E antitoxin. Because small catfish are often unavailable, the utility of adult zebrafish (Danio rerio) was evaluated in BoNT/E and VTC bioassays. Channel catfish and zebrafish susceptibilities were compared using trypsin-activated BoNT/E in a 96-hr trial by intracoelomically administering 0, 1.87, 3.7, 7.5, 15, or 30 pg of toxin per gram of body weight (g-bw) of fish. All of the zebrafish died at the 7.5 pg/g-bw and higher, while the catfish died at the 15 pg/g-bw dose and higher. To test the bioassay, sera from VTC-affected fish or control sera were intracoelomically injected at a dose of 10 µl per zebrafish and 20 µl/g-bw for channel catfish. At 96 hr post-injection, 78% of the zebrafish and 50% of the catfish receiving VTC sera died, while no control fish died. When the VTC sera were preincubated with BoNT/E antitoxin, they became nontoxic to zebrafish. Histology of zebrafish injected with either VTC serum or BoNT/E demonstrated renal necrosis. Normal catfish serum was toxic to larval zebrafish in immersion exposures, abrogating their utility in VTC bioassays. The results demonstrate bioassays using adult zebrafish for detecting BoNT/E and VTC are sensitive and practical.

  20. INDUCED AND SPONTANEOUS NEOPLASIA IN ZEBRAFISH.

    EPA Science Inventory

    To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors, and to compare zebrafish tumors with human tumors. To determine whether the commonly-used germ line mutagen, ethylnitrosourea (ENU) induces tumors, we ...

  1. INDUCED AND SPONTANEOUS NEOPLASIA IN ZEBRAFISH.

    EPA Science Inventory

    To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors, and to compare zebrafish tumors with human tumors. To determine whether the commonly-used germ line mutagen, ethylnitrosourea (ENU) induces tumors, we ...

  2. The genetic basis of cardiac function: dissection by zebrafish (Danio rerio) screens.

    PubMed Central

    Warren, K S; Wu, J C; Pinet, F; Fishman, M C

    2000-01-01

    The vertebrate heart differs from chordate ancestors both structurally and functionally. Genetic units of form, termed 'modules', are identifiable by mutation, both in zebrafish and mouse, and correspond to features recently acquired in evolution, such as the ventricular chamber or endothelial lining of the vessels and heart. Zebrafish (Danio rerio) genetic screens have provided a reasonably inclusive set of such genes. Normal cardiac function may also be disrupted by single-gene mutations in zebrafish. Individual mutations may perturb contractility or rhythm generation. The zebrafish mutations which principally disturb cardiac contractility fall into two broad phenotypic categories, 'dilated' and 'hypertrophic'. Interestingly, these correspond to the two primary types of heart failure in humans. These disorders of early cardiac function provide candidate genes to be examined in complex human heart diseases, including arrhythmias and heart failure. PMID:11128987

  3. Zebrafish as an experimental model: strategies for developmental and molecular neurobiology studies.

    PubMed

    Key, Brian; Devine, Christine A

    2003-01-01

    Zebrafish provide a rapid and effective means for assessing gene function in the vertebrate nervous system. By employing gain- and loss-of-function techniques it is possible to obtain insights into the roles of both wild-type and heterologously expressed genes. Such approaches enable rapid progression from gene discovery to gene expression and finally to gene function even when examining development of a tissue as complex as the nervous system. Exploiting the full potential of zebrafish as a bioassay for the nervous system will require, not only an understanding of the molecular and cellular basis of normal zebrafish development, but also an appreciation of comparative processes in other species. When applied to mutant animals, classic morphological approaches and contemporary molecular genetic techniques are providing a wealth of information on the development of the nervous system at the molecular, cell, system and behavioural levels. Zebrafish are now emerging as an important tool, supporting mouse genetical approaches for understanding neural function in vertebrates.

  4. Cloning, expression and functional study of translation elongation factor 2 (EF-2) in zebrafish.

    PubMed

    Zhang, Shu-Hong; Yao, Ji-Hua; Song, Huai-Dong; Wang, Lu; Xue, Jing-Lun

    2006-01-01

    We have identified translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of the DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame between nucleotide 55 to 2631 and encodes a protein of 858 amino acids. Zebrafish EF-2 protein shares 92%, 93%, 93% and 92% identity with the corresponding amino acid sequence in human, mouse, Chinese hamster and Gallus EF-2, respectively. Whole-mount in situ hybridization showed that zebrafish EF-2 was a developmentally regulated gene and might play important roles during the early development of zebrafish embryos. Therefore, we further studied the function of EF-2 during early embryogenesis. Using morpholino antisense oligo knockdown assays, anti-MO injected embryos were found to display abnormal development. The yolk balls were larger than normal and the melanophores spreading on their bodies became fewer. Furthermore, their tails were incurvate and their lenses were much smaller than those of the normal embryos. However the EF-2 overexpression data showed that extra EF-2 protein had no obvious effect on zebrafish embryonic development.

  5. Zebrafish antipredatory responses: A future for translational research?

    PubMed Central

    Gerlai, Robert

    2011-01-01

    Human neuropsychiatric conditions associated with abnormally exaggerated or misdirected fear (anxiety disorders and phobias) still represent a large unmet medical need because the biological mechanisms underlying these diseases are not well understood. Animal models have been proposed to facilitate this research. Here I review the literature with a focus on zebrafish, an upcoming laboratory organism in behavioral brain research. I argue that abnormal human fear responses are likely the result of the malfunction of neurobiological mechanisms (brain areas, circuits and/or molecular mechanisms) that originally evolved to support avoidance of predators or other harm in nature. I also argue that the understanding of the normal as well as pathological functioning of such mechanisms may be best achieved if one utilizes naturalistic experimental approaches. In case of laboratory model organisms, this may entail presenting stimuli associated with predators and measuring species-specific antipredatory responses. Although zebrafish is a relatively new subject of such inquiry, I review the recently rapidly increasing number of zebrafish studies in this area, and conclude that zebrafish is a promising research tool for the analysis of the neurobiology and genetics of vertebrate fear responses. PMID:19836422

  6. Retinoic acid expands the evolutionarily reduced dentition of zebrafish

    PubMed Central

    Seritrakul, Pawat; Samarut, Eric; Lama, Tenzing T. S.; Gibert, Yann; Laudet, Vincent; Jackman, William R.

    2012-01-01

    Zebrafish lost anterior teeth during evolution but retain a posterior pharyngeal dentition that requires retinoic acid (RA) cell-cell signaling for its development. The purposes of this study were to test the sufficiency of RA to induce tooth development and to assess its role in evolution. We found that exposure of embryos to exogenous RA induces a dramatic anterior expansion of the number of pharyngeal teeth that later form and shifts anteriorly the expression patterns of genes normally expressed in the posterior tooth-forming region, such as pitx2 and dlx2b. After RA exposure, we also observed a correlation between cartilage malformations and ectopic tooth induction, as well as abnormal cranial neural crest marker gene expression. Additionally, we observed that the RA-induced zebrafish anterior teeth resemble in pattern and number the dentition of fish species that retain anterior pharyngeal teeth such as medaka but that medaka do not express the aldh1a2 RA-synthesizing enzyme in tooth-forming regions. We conclude that RA is sufficient to induce anterior ectopic tooth development in zebrafish where teeth were lost in evolution, potentially by altering neural crest cell development, and that changes in the location of RA synthesis correlate with evolutionary changes in vertebrate dentitions.—Seritrakul, P., Samarut, E., Lama, T. T. S., Gibert, Y., Laudet, V., Jackman, W. R. Retinoic acid expands the evolutionarily reduced dentition of zebrafish. PMID:22942074

  7. Structural characterization of glycosaminoglycans from zebrafish in different ages

    PubMed Central

    Zhang, Fuming; Zhang, Zhenqing; Thistle, Robert; McKeen, Lindsey; Hosoyama, Saori; Toida, Toshihiko

    2009-01-01

    The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using 1H-NMR. PMID:18777207

  8. Early Retinoic acid deprivation in developing zebrafish results in microphthalmia

    PubMed Central

    Le, Hong-Gam T.; Dowling, John E.; Cameron, D. Joshua

    2013-01-01

    Vitamin A deficiency causes impaired vision and blindness in millions of children around the world. Previous studies in zebrafish have demonstrated that retinoic acid (RA), the acid form of vitamin A, plays a vital role in early eye development. The objective of this study was to describe the effects of early RA deficiency by treating zebrafish with diethylaminobenzaldehyde (DEAB), a potent inhibitor of the enzyme retinaldehyde dehydrogenase (Raldh) that converts retinal to RA. Zebrafish embryos were treated for 2 hours beginning at 9 hours post-fertilization (hpf). Gross morphology and retinal development were examined at regular intervals for 5 days after treatment. The optokinetic reflex (OKR) test, visual background adaptation (VBA) test, and the electroretinogram (ERG) were performed to assess visual function and behavior. Early treatment of zebrafish embryos with 100 μM DEAB (9hr) resulted in reduced eye size and this microphthalmia persisted through larval development. Retinal histology revealed that DEAB eyes, had significant developmental abnormalities but had relatively normal retinal lamination by 5.5 days post-fertilization (dpf). However, the fish showed neither, an OKR or VBA response. Further, the retina did not respond to light as measured by the ERG. We conclude that early deficiency of RA during eye development causes microphthalmia as well as other visual defects, and that timing of the RA deficiency is critical to the developmental outcome. PMID:23013828

  9. Effects of metal ions on cyprinid fish immune response: In vitro effects of Zn2/sup +/ and Mn/sup 2+/ on the mitogenic response of carp pronephros lymphocytes

    SciTech Connect

    Ghanmi, Z.; Rouabhia, M.; Othmane, O.; Deschaux, P.A.

    1989-04-01

    Lymphocytes from the pronephros of carp (Cyprinus carpio L) have been subjected to transformation by mitogens, concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharides (LPS), with Zn or Mn at varying concentrations. Addition of Zn/sup 2+/ (10(-7) to 10(-3) M) to mitogen-stimulated T and B cells enhanced (/sup 3/H)thymidine incorporation. Addition of 10(-5) M Zn/sup 2+/ inhibited the response to Con A, PHA, and LPS. At this concentration, Zn was toxic. Addition of Mn2+ (10(-7) to 10(-3) M) to mitogen-stimulated lymphocytes enhanced (/sup 3/H)thymidine incorporation. This effect was observed with Con A- and PHA-stimulated lymphocytes, but not with LPS-stimulated lymphocytes. In contrast, addition of 10(-1) M Mn/sup 2+/ to lymphocyte cultures exerted an inhibitor on the response to Con A or to PHA, while the response to LPS was unaffected. Addition of 10(-1) M Mn/sup 2+/ to Con A- or PHA-stimulated cultures at different times after initiation of stimulation indicated that Mn/sup 2+/ was inhibitory only when it was added before the first 16 hr of cultures. The inhibition induced by 10(-1) M Mn2+ could be reversed by adding 2 mM CaCl/sub 2/ to the culture.

  10. Cadherin-6 Function in Zebrafish Retinal Development

    PubMed Central

    Liu, Qin; Londraville, Richard; Marrs, James A.; Wilson, Amy L.; Mbimba, Thomas; Murakami, Tohru; Kubota, Fumitaka; Zheng, Weiping; Fatkins, David G.

    2008-01-01

    Cadherin cell adhesion molecules play crucial roles in vertebrate development including the development of the visual system. Most studies have focused on examining functions of classical type I cadherins (e.g. cadherin-2) in visual system development. There is little information on the function of classical type II cadherins (e.g. cadherin-6) in the development of the vertebrate visual system. To gain insight into cadherin-6 role in the formation of the retina, we analyzed differentiation of retinal ganglion cells, amacrine cells and photoreceptors in zebrafish embryos injected with cadherin-6 specific antisense morpholino oligonucleotides. Differentiation of the retinal neurons in cadherin-6 knockdown embryos (cdh6 morphants) was analyzed using multiple markers. We found that expression of transcription factors important for retinal development was greatly reduced, and expression of Notch-Delta genes and proneural gene ath5 was altered in the cdh6 morphant retina. The retinal lamination was present in the morphants, although the morphant eyes were significantly smaller than control embryos due mainly to decreased cell proliferation. Differentiation of the retinal ganglion cells, amacrine cells and photoreceptors was severely disrupted in the cdh6 morphants due to a significant delay in neuronal differentiation. Our results suggest that cadherin-6 plays an important role in the normal formation of the zebrafish retina. PMID:18506771

  11. Maintenance of Zebrafish Lines at the European Zebrafish Resource Center

    PubMed Central

    Borel, Nadine; Ferg, Marco; Maier, Jana Viktoria; Strähle, Uwe

    2016-01-01

    Abstract We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections. PMID:27351617

  12. Zebrafish Sensitivity to Botulinum Neurotoxins

    PubMed Central

    Chatla, Kamalakar; Gaunt, Patricia S.; Petrie-Hanson, Lora; Ford, Lorelei; Hanson, Larry A.

    2016-01-01

    Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins. PMID:27153088

  13. The Zebrafish Neurophenome Database (ZND): a dynamic open-access resource for zebrafish neurophenotypic data.

    PubMed

    Kyzar, Evan; Zapolsky, Ivan; Green, Jeremy; Gaikwad, Siddharth; Pham, Mimi; Collins, Christopher; Roth, Andrew; Stewart, Adam Michael; St-Pierre, Paul; Hirons, Budd; Kalueff, Allan V

    2012-03-01

    Zebrafish (Danio rerio) are widely used in neuroscience research, where their utility as a model organism is rapidly expanding. Low cost, ease of experimental manipulations, and sufficient behavioral complexity make zebrafish a valuable tool for high-throughput studies in biomedicine. To complement the available repositories for zebrafish genetic information, there is a growing need for the collection of zebrafish neurobehavioral and neurological phenotypes. For this, we are establishing the Zebrafish Neurophenome Database (ZND; www.tulane.edu/∼znpindex/search ) as a new dynamic online open-access data repository for behavioral and related physiological data. ZND, currently focusing on adult zebrafish, combines zebrafish neurophenotypic data with a simple, easily searchable user interface, which allow scientists to view and compare results obtained by other laboratories using various treatments in different testing paradigms. As a developing community effort, ZND is expected to foster innovative research using zebrafish by federating the growing body of zebrafish neurophenotypic data.

  14. Neurochemical measurements in the zebrafish brain

    PubMed Central

    Jones, Lauren J.; McCutcheon, James E.; Young, Andrew M. J.; Norton, William H. J.

    2015-01-01

    The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain. PMID:26441575

  15. Mapping of zebrafish research: a global outlook.

    PubMed

    Kinth, Priyamvadah; Mahesh, Gopalakrishnan; Panwar, Yatish

    2013-12-01

    On the basis of analysis of 17,151 records on zebrafish identified from Zebrafish Information Network: the zebrafish model organism database and Web of Science, the research performance on this model organism has been evaluated. The earliest research work on zebrafish as reflected in the databases goes back to 1951. After a rather slow growth till the 1980s, research on zebrafish gained momentum in the 1990s. Analysis shows a rapid and consistent increase in the publication output with 226 publications in the year 1996, to 1929 publications in the year 2012. The prominent areas of zebrafish research, journals, and leading authors as reflected from the research output have been identified. USA is the most productive country with 8196 articles. The most frequently used keywords were also determined to gain insights about the research trends and some of the commonly used keywords other than zebrafish and Danio rerio are development, retina, and gene expression.

  16. Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio).

    PubMed

    Jianjie, Chen; Wenjuan, Xue; Jinling, Cao; Jie, Song; Ruhui, Jia; Meiyan, Li

    2016-02-01

    Excessive fluoride in natural water ecosystem has the potential to detrimentally affect thyroid endocrine system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of fluoride on growth performance, thyroid histopathology, thyroid hormone levels, and gene expressions in the HPT axis in male zebrafish (Danio rerio) exposed to different determined concentrations of 0.1, 0.9, 2.0 and 4.1 M of fluoride to investigate the effects of fluoride on thyroid endocrine system and the potential toxic mechanisms caused by fluoride. The results indicated that the growth of the male zebrafish used in the experiments was significantly inhibited, the thyroid microtrastructure was changed, and the levels of T3 and T4 were disturbed in fluoride-exposed male fish. In addition, the expressional profiles of genes in HPT axis displayed alteration. The expressions of all studied genes were significantly increased in all fluoride-exposed male fish after exposure for 45 days. The transcriptional levels of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSH), thyroglobulin (TG), sodium iodide symporter (NIS), iodothyronine I (DIO1), and thyroid hormone receptor alpha (TRα) were also elevated in all fluoride-exposed male fish after 90 days of exposure, while the inconsistent expressions were found in the mRNA of iodothyronineⅡ (DIO2), UDP glucuronosyltransferase 1 family a, b (UGT1ab), transthyretin (TTR), and thyroid hormone receptor beta (TRβ). These results demonstrated that fluoride could notably inhibit the growth of zebrafish, and significantly affect thyroid endocrine system by changing the microtrastructure of thyroid, altering thyroid hormone levels and endocrine-related gene expressions in male zebrafish. All above indicated that fluoride could pose a great threat to thyroid endocrine system, thus detrimentally affected the normal function of thyroid of male zebrafish. Copyright © 2015

  17. Husbandry of zebrafish, Danio rerio, and the cortisol stress response.

    PubMed

    Pavlidis, Michail; Digka, Nikoletta; Theodoridi, Antonia; Campo, Aurora; Barsakis, Konstantinos; Skouradakis, Gregoris; Samaras, Athanasios; Tsalafouta, Alexandra

    2013-12-01

    The effect of common husbandry conditions (crowding, social environment, water quality, handling, and background color) on the cortisol stress response in adult zebrafish, Danio rerio, was investigated to check the usefulness of zebrafish as a model organism in aquaculture research. In addition, a noninvasive methodology for assessing stress was evaluated. Zebrafish showed a fast cortisol response with high values at 30 min that returned to basal levels within 2 h of poststress. There was a significant positive correlation between trunk cortisol concentrations and the free water cortisol rate (r(2)=0.829-0.850, p<0.001), indicating that measurement of the water-borne cortisol release rate may serve as a noninvasive and reliable stress indicator at the population level. Crowding resulted in 13- to 21-fold greater mean trunk cortisol concentrations compared with controls. However, even at low stocking density (2-5 fish/L), the maintenance cost was higher than the one at higher densities (10 fish/L) due to the formation of dominance hierarchies. The background color affected trunk cortisol concentrations, with fish exposed to brighter backgrounds (green and white) showing 3- to 8-fold greater mean trunk cortisol concentrations than fish exposed to a black background or transparent aquaria. Fish exposed to high stocking densities for 2 h or 5 days had similar high mean trunk cortisol levels, indicating that exposure of fish for the period of 2 h to a specific stressor may represent a chronic situation in zebrafish. It is concluded that adult laboratory zebrafish had a preference for a transparent or black background aquarium, at a number of 10 individuals per 2 L of available water volume, to express their normal behavior and avoid increased cortisol stress reaction.

  18. Object recognition memory in zebrafish.

    PubMed

    May, Zacnicte; Morrill, Adam; Holcombe, Adam; Johnston, Travis; Gallup, Joshua; Fouad, Karim; Schalomon, Melike; Hamilton, Trevor James

    2016-01-01

    The novel object recognition, or novel-object preference (NOP) test is employed to assess recognition memory in a variety of organisms. The subject is exposed to two identical objects, then after a delay, it is placed back in the original environment containing one of the original objects and a novel object. If the subject spends more time exploring one object, this can be interpreted as memory retention. To date, this test has not been fully explored in zebrafish (Danio rerio). Zebrafish possess recognition memory for simple 2- and 3-dimensional geometrical shapes, yet it is unknown if this translates to complex 3-dimensional objects. In this study we evaluated recognition memory in zebrafish using complex objects of different sizes. Contrary to rodents, zebrafish preferentially explored familiar over novel objects. Familiarity preference disappeared after delays of 5 mins. Leopard danios, another strain of D. rerio, also preferred the familiar object after a 1 min delay. Object preference could be re-established in zebra danios by administration of nicotine tartrate salt (50mg/L) prior to stimuli presentation, suggesting a memory-enhancing effect of nicotine. Additionally, exploration biases were present only when the objects were of intermediate size (2 × 5 cm). Our results demonstrate zebra and leopard danios have recognition memory, and that low nicotine doses can improve this memory type in zebra danios. However, exploration biases, from which memory is inferred, depend on object size. These findings suggest zebrafish ecology might influence object preference, as zebrafish neophobia could reflect natural anti-predatory behaviour. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Modular organization of axial microcircuits in zebrafish

    PubMed Central

    Bagnall, Martha W.; McLean, David L.

    2014-01-01

    Locomotion requires precise control of spinal networks. In tetrapods and bipeds, dynamic regulation of locomotion is simplified by the modular organization of spinal limb circuits, but it is not known whether their predecessors, fish axial circuits, are similarly organized. Here, we demonstrate that the larval zebrafish spinal cord contains distinct, parallel microcircuits for independent control of dorsal and ventral musculature on each side of the body. During normal swimming, dorsal and ventral microcircuits are equally active; but during postural correction, fish differentially engage these microcircuits to generate torque for self-righting. These findings reveal greater complexity in the axial spinal networks responsible for swimming than previously recognized and suggest an early template of modular organization for more complex locomotor circuits in later vertebrates. PMID:24408436

  20. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    PubMed Central

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-01-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model. PMID:26135470

  1. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-07-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model.

  2. Microsporidiosis in Zebrafish Research Facilities

    PubMed Central

    Sanders, Justin L.; Watral, Virginia; Kent, Michael L.

    2014-01-01

    Pseudoloma neurophilia (Microsporidia) is the most common pathogen detected in zebrafish (Danio rerio) from research facilities. The parasite infects the central nervous system and muscle and may be associated with emaciation and skeletal deformities. However, many fish exhibit sub-clinical infections. Another microsporidium, Pleistophora hyphessobryconis, has recently been detected in a few zebrafish facilities. Here, we review the methods for diagnosis and detection, modes of transmission, and approaches used to control microsporidia in zebrafish, focusing on P. neurophilia. The parasite can be readily transmitted by feeding spores or infected tissues, and we show that cohabitation with infected fish is also an effective means of transmission. Spores are released from live fish in various manners, including through the urine, feces, and sex products during spawning. Indeed, P. neurophilia infects both the eggs and ovarian tissues, where we found concentrations ranging from 12,000 to 88,000 spores per ovary. Hence, various lines of evidence support the conclusion that maternal transmission is a route of infection: spores are numerous in ovaries and developing follicles in infected females, spores are present in spawned eggs and water from spawning tanks based on polymerase chain reaction tests, and larvae are very susceptible to the infection. Furthermore, egg surface disinfectants presently used in zebrafish laboratories are ineffective against microsporidian spores. At this time, the most effective method for prevention of these parasites is avoidance. PMID:23382342

  3. Contextual fear conditioning in zebrafish.

    PubMed

    Kenney, Justin W; Scott, Ian C; Josselyn, Sheena A; Frankland, Paul W

    2017-10-01

    Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development of a contextual fear conditioning paradigm using an electric shock as the aversive stimulus. We find that contextual fear conditioning is modulated by shock intensity, prevented by an established amnestic agent (MK-801), lasts at least 14 d, and exhibits extinction. Furthermore, fish of various background strains (AB, Tu, and TL) are able to acquire fear conditioning, but differ in fear extinction rates. Taken together, we find that contextual fear conditioning in zebrafish shares many similarities with the widely used contextual fear conditioning paradigm in rodents. Combined with the amenability of genetic manipulation in zebrafish, we anticipate that our paradigm will prove to be a useful complementary system in which to examine the molecular basis of vertebrate learning and memory. © 2017 Kenney et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Zebrafish sex: a complicated affair

    PubMed Central

    Liew, Woei Chang

    2014-01-01

    In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future. PMID:24148942

  5. Precise Editing of the Zebrafish Genome Made Simple and Efficient

    PubMed Central

    Hoshijima, Kazuyuki; Jurynec, Michael J.; Grunwald, David Jonah

    2016-01-01

    SUMMARY We present simple and efficient methods for creating heritable modifications of the zebrafish genome. Precisely modified alleles are generated by homologous recombination between the host genome and dsDNA donor molecules, stimulated by the induction of chromosomally targeted DSBs. Several kilobase-long tracts of genome sequence can be replaced. Tagging donor sequences with reporter genes that can be subsequently excised improves recovery of edited alleles by an order of magnitude and facilitates recovery of recessive and phenotypically silent conditional mutations. We generate and demonstrate functionality of: i) alleles with a single codon change, ii) an allele encoding an epitope-tagged version of an endogenous protein, iii) alleles expressing reporter proteins, and iv) a conditional allele in which an exon is flanked by recombinogenic loxP sites. Our methods make recovery of a broad range of genome editing events very practicable, significantly advancing applicability of the zebrafish for studying normal biological processes and modeling diseases. PMID:27003937

  6. Behavioural fever in zebrafish larvae.

    PubMed

    Rey, Sonia; Moiche, Visila; Boltaña, Sebastian; Teles, Mariana; MacKenzie, Simon

    2017-02-01

    Behavioural fever has been reported in different species of mobile ectotherms including the zebrafish, Danio rerio, in response to exogenous pyrogens. In this study we report, to our knowledge for the first time, upon the ontogenic onset of behavioural fever in zebrafish (Danio rerio) larvae. For this, zebrafish larvae (from first feeding to juveniles) were placed in a continuous thermal gradient providing the opportunity to select their preferred temperature. The novel thermal preference aquarium was based upon a continuous vertical column system and allows for non-invasive observation of larvae vertical distribution under isothermal (TR at 28 °C) and thermal gradient conditions (TCH: 28-32 °C). Larval thermal preference was assessed under both conditions with or without an immersion challenge, in order to detect the onset of the behavioural fever response. Our results defined the onset of the dsRNA induced behavioural fever at 18-20 days post fertilization (dpf). Significant differences were observed in dsRNA challenged larvae, which prefer higher temperatures (1-4 °C increase) throughout the experimental period as compared to non-challenged larvae. In parallel we measured the abundance of antiviral transcripts; viperin, gig2, irf7, trim25 and Mxb mRNAs in dsRNA challenged larvae under both thermal regimes: TR and TCh. Significant increases in the abundance of all measured transcripts were recorded under thermal choice conditions signifying that thermo-coupling and the resultant enhancement of the immune response to dsRNA challenge occurs from 18 dpf onwards in the zebrafish. The results are of importance as they identify a key developmental stage where the neuro-immune interface matures in the zebrafish likely providing increased resistance to viral infection.

  7. Development of social behavior in young zebrafish.

    PubMed

    Dreosti, Elena; Lopes, Gonçalo; Kampff, Adam R; Wilson, Stephen W

    2015-01-01

    Adult zebrafish are robustly social animals whereas larva is not. We designed an assay to determine at what stage of development zebrafish begin to interact with and prefer other fish. One week old zebrafish do not show significant social preference whereas most 3 weeks old zebrafish strongly prefer to remain in a compartment where they can view conspecifics. However, for some individuals, the presence of conspecifics drives avoidance instead of attraction. Social preference is dependent on vision and requires viewing fish of a similar age/size. In addition, over the same 1-3 weeks period larval zebrafish increasingly tend to coordinate their movements, a simple form of social interaction. Finally, social preference and coupled interactions are differentially modified by an NMDAR antagonist and acute exposure to ethanol, both of which are known to alter social behavior in adult zebrafish.

  8. Glial Cell Development and Function in Zebrafish

    PubMed Central

    Lyons, David A.; Talbot, William S.

    2015-01-01

    The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function. PMID:25395296

  9. Development of social behavior in young zebrafish

    PubMed Central

    Dreosti, Elena; Lopes, Gonçalo; Kampff, Adam R.; Wilson, Stephen W.

    2015-01-01

    Adult zebrafish are robustly social animals whereas larva is not. We designed an assay to determine at what stage of development zebrafish begin to interact with and prefer other fish. One week old zebrafish do not show significant social preference whereas most 3 weeks old zebrafish strongly prefer to remain in a compartment where they can view conspecifics. However, for some individuals, the presence of conspecifics drives avoidance instead of attraction. Social preference is dependent on vision and requires viewing fish of a similar age/size. In addition, over the same 1–3 weeks period larval zebrafish increasingly tend to coordinate their movements, a simple form of social interaction. Finally, social preference and coupled interactions are differentially modified by an NMDAR antagonist and acute exposure to ethanol, both of which are known to alter social behavior in adult zebrafish. PMID:26347614

  10. Whole-body imaging of a hypercholesterolemic female zebrafish by using synchrotron X-ray micro-CT.

    PubMed

    Seo, Eunseok; Lim, Jae-Hong; Seo, Seung Jun; Lee, Sang Joon

    2015-02-01

    Zebrafish has been used as a powerful model system in biological and biomedical studies studying development and diseases. Comparative, functional, and developmental studies on zebrafish morphology require precise visualization of 3D morphological structures. Few methods that can visualize whole-volume of zebrafish tissues are available because optical bio-imaging methods are limited by pigmentation and hard tissues. To overcome these limitations, the 3D microstructures of a hypercholesterolemic zebrafish model are visualized using synchrotron X-ray micro-computed tomography (SR-μCT). The model spatial resolution ranged from sub- to several microns. The microstructures of various zebrafish organs are observed by combining high-contrast staining (osmium tetroxide and uranyl acetate) and embedding a protocol to enhance the image contrast of soft tissues. Furthermore, blood vessels are identified using a barium sulfate injection technique. The internal organs and cells, such as liver, intestine, oocytes, and adipocytes, of a hypercholesterolemic zebrafish are compared with those of normal organs and cells. The SR-μCT is useful for understanding the pathogenesis of circulatory vascular diseases by detecting the modifications in the 3D morphological structures of the whole body of the zebrafish. This bio-imaging technique can be readily used to study other disease models.

  11. Whole-Body Imaging of a Hypercholesterolemic Female Zebrafish by Using Synchrotron X-Ray Micro-CT

    PubMed Central

    Seo, Eunseok; Lim, Jae-Hong; Seo, Seung Jun

    2015-01-01

    Abstract Zebrafish has been used as a powerful model system in biological and biomedical studies studying development and diseases. Comparative, functional, and developmental studies on zebrafish morphology require precise visualization of 3D morphological structures. Few methods that can visualize whole-volume of zebrafish tissues are available because optical bio-imaging methods are limited by pigmentation and hard tissues. To overcome these limitations, the 3D microstructures of a hypercholesterolemic zebrafish model are visualized using synchrotron X-ray micro-computed tomography (SR-μCT). The model spatial resolution ranged from sub- to several microns. The microstructures of various zebrafish organs are observed by combining high-contrast staining (osmium tetroxide and uranyl acetate) and embedding a protocol to enhance the image contrast of soft tissues. Furthermore, blood vessels are identified using a barium sulfate injection technique. The internal organs and cells, such as liver, intestine, oocytes, and adipocytes, of a hypercholesterolemic zebrafish are compared with those of normal organs and cells. The SR-μCT is useful for understanding the pathogenesis of circulatory vascular diseases by detecting the modifications in the 3D morphological structures of the whole body of the zebrafish. This bio-imaging technique can be readily used to study other disease models. PMID:25521241

  12. Zona Pellucida Domain-Containing Protein β-Tectorin is Crucial for Zebrafish Proper Inner Ear Development

    PubMed Central

    Yang, Chung-Hsiang; Cheng, Chia-Hsiung; Chen, Gen-Der; Liao, Wei-Hao; Chen, Yi-Chung; Huang, Kai-Yun; Hwang, Pung-Pung; Hwang, Sheng-Ping L.; Huang, Chang-Jen

    2011-01-01

    Background The zona pellucida (ZP) domain is part of many extracellular proteins with diverse functions from structural components to receptors. The mammalian β-tectorin is a protein of 336 amino acid residues containing a single ZP domain and a putative signal peptide at the N-terminus of the protein. It is 1 component of a gel-like structure called the tectorial membrane which is involved in transforming sound waves into neuronal signals and is important for normal auditory function. β-Tectorin is specifically expressed in the mammalian and avian inner ear. Methodology/Principal Findings We identified and cloned the gene encoding zebrafish β-tectorin. Through whole-mount in situ hybridization, we demonstrated that β-tectorin messenger RNA was expressed in the otic placode and specialized sensory patch of the inner ear during zebrafish embryonic stages. Morpholino knockdown of zebrafish β-tectorin affected the position and number of otoliths in the ears of morphants. Finally, swimming behaviors of β-tectorin morphants were abnormal since the development of the inner ear was compromised. Conclusions/Significance Our results reveal that zebrafish β-tectorin is specifically expressed in the zebrafish inner ear, and is important for regulating the development of the zebrafish inner ear. Lack of zebrafish β-tectorin caused severe defects in inner ear formation of otoliths and function. PMID:21829695

  13. Protective Role of Comfrey Leave Extracts on UV-induced Zebrafish Fin Damage

    PubMed Central

    Cheng, Chien-Chung; Chou, Chi-Yuan; Chang, Yao-Chin; Wang, Hsuan-Wen; Wen, Chi-Chung; Chen, Yau-Hung

    2014-01-01

    In zebrafish, UV exposure leads to fin malformation phenotypes including fin reduction or absence. The present study evaluated UV-protective activities of comfrey leaves extracts in a zebrafish model by recording fin morphological changes. Chemopreventive effects of comfrey leave extracts were evaluated using Kaplan-Meier analysis and Cox proportional hazards regression. The results showed that (1) the mean times of return to normal fin in the UV+comfrey (50 and 100 ppm) groups were 3.43 and 2.86 days and were quicker compared with that in the UV only group (4.21 days); (2) zebrafish fins in the UV+comfrey (50 and 100 ppm) groups were 2.05 and 3.25 times more likely to return to normal than those in the UV only group; and (3) comfrey leave extracts had UV-absorbance abilities and significantly reduced ROS production in UV-exposed zebrafish embryos, which may attenuate UV-mediated apoptosis. In conclusion, comfrey leaves extracts may have the potential to be developed as UV-protective agents to protect zebrafish embryos from UV-induced damage. PMID:25352712

  14. Zebrafish Social Behavior in the Wild.

    PubMed

    Suriyampola, Piyumika S; Shelton, Delia S; Shukla, Rohitashva; Roy, Tamal; Bhat, Anuradha; Martins, Emília P

    2016-02-01

    Wild zebrafish exhibit a wide range of behavior. We found abundant wild zebrafish in flowing rivers and still water, in large, tightly-knit groups of hundreds of individuals, as well as in small, loose shoals. In two still-water populations, zebrafish were quite small in body size, common, and in tight groups of up to 22 fish. As in earlier laboratory studies, these zebrafish exhibited very low levels of aggression. In slowly flowing water in central India, zebrafish were relatively rare and gathered in small shoals (4-12 fish), often with other small fish, such as Rasbora daniconius. These stream zebrafish were larger in body size (27 mm TL) and much more aggressive than those in still water. In a second river population with much faster flowing water, zebrafish were abundant and again relatively large (21 mm TL). These zebrafish occurred in very large (up to 300 individuals) and tightly-knit (nearest-neighbor distances up to 21 mm) groups that exhibited collective rheotaxis and almost no aggression. This remarkable variation in social behavior of wild zebrafish offers an opportunity for future studies of behavioral genetics, development, and neuroscience.

  15. A critical period for functional vestibular development in zebrafish

    NASA Technical Reports Server (NTRS)

    Moorman, Stephen J.; Cordova, Rodolfo; Davies, Sarah A.

    2002-01-01

    We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe

  16. A critical period for functional vestibular development in zebrafish

    NASA Technical Reports Server (NTRS)

    Moorman, Stephen J.; Cordova, Rodolfo; Davies, Sarah A.

    2002-01-01

    We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe

  17. Identification and functional characterization of zebrafish solute carrier Slc16a2 (Mct8) as a thyroid hormone membrane transporter.

    PubMed

    Arjona, Francisco J; de Vrieze, Erik; Visser, Theo J; Flik, Gert; Klaren, Peter H M

    2011-12-01

    Most components of the thyroid system in bony fish have been described and characterized, with the notable exception of thyroid hormone membrane transporters. We have cloned, sequenced, and expressed the zebrafish solute carrier Slc16a2 (also named monocarboxylate transporter Mct8) cDNA and established its role as a thyroid hormone transport protein. The cloned cDNA shares 56-57% homology with its mammalian orthologs. The 526-amino-acid sequence contains 12 predicted transmembrane domains. An intracellular N-terminal PEST domain, thought to be involved in proteolytic processing of the protein, is present in the zebrafish sequence. Measured at initial rate and at the body/rearing temperature of zebrafish (26 C), T(3) uptake by zebrafish Slc16a2 is a saturable process with a calculated Michaelis-Menten constant of 0.8 μM T(3). The rate of T(3) uptake is temperature dependent and Na(+) independent. Interestingly, at 26 C, zebrafish Slc16a2 does not transport T(4). This implies that at a normal body temperature in zebrafish, Slc16a2 protein is predominantly involved in T(3) uptake. When measured at 37 C, zebrafish Slc16a2 transports T(4) in a Na(+)-independent manner. In adult zebrafish, the Slc16a2 gene is highly expressed in brain, gills, pancreas, liver, pituitary, heart, kidney, and gut. Beginning from the midblastula stage, Slc16a2 is also expressed during zebrafish early development, the highest expression levels occurring 48 h after fertilization. This is the first direct evidence for thyroid hormone membrane transporters in fish. We suggest that Slc16a2 plays a key role in the local availability of T(3) in adult tissues as well as during the completion of morphogenesis of primary organ systems.

  18. Knockdown of Tmem234 in zebrafish results in proteinuria.

    PubMed

    Rodriguez, Patricia Q; Oddsson, Asmundur; Ebarasi, Lwaki; He, Bing; Hultenby, Kjell; Wernerson, Annika; Betsholtz, Christer; Tryggvason, Karl; Patrakka, Jaakko

    2015-12-01

    Podocytes are highly specialized epithelial cells located at the outer aspects of the glomerular capillary tuft and critical components of the kidney filtration barrier. To maintain their unique features, podocytes express a number of proteins that are only sparsely found elsewhere in the body. In this study, we have identified four (Tmem234, Znf185, Lrrc49, and Slfn5) new highly podocyte-enriched proteins. The proteins are strongly expressed by podocytes, while other parts of the kidney show only weak or no expression. Tmem234, Slfn5, and Lrrc49 are located in foot processes, whereas Znf185 is found in both foot and major processes. Expressional studies in developing kidneys show that these proteins are first expressed at the capillary stage glomerulus, the same stage when the formation of major and foot processes begins. We identified zebrafish orthologs for Tmem234 and Znf185 genes and knocked down their expression using morpholino technology. Studies in zebrafish larvae indicate that Tmem234 is essential for the organization and functional integrity of the pronephric glomerulus filtration barrier, as inactivation of Tmem234 expression results in foot process effacement and proteinuria. In summary, we have identified four novel highly podocyte-enriched proteins and show that one of them, Tmem234, is essential for the normal filtration barrier in the zebrafish pronephric glomerulus. Identification of new molecular components of the kidney filtration barrier opens up possibilities to study their role in glomerulus biology and diseases. Copyright © 2015 the American Physiological Society.

  19. Zebrafish atoh8 mutants do not recapitulate morpholino phenotypes

    PubMed Central

    Place, Elsie S.; Smith, James C.

    2017-01-01

    Atoh8 is a bHLH transcription factor expressed in pancreas, skeletal muscle, the nervous system, and cardiovascular tissues during embryological development. Although it has been implicated in the regulation of pancreatic and endothelial cell differentiation, the phenotypic consequences of Atoh8 loss are uncertain. Conclusions from knockout studies in the mouse differ widely depending on the targeting strategy used, while atoh8 knockdown by interfering morpholino oligonucleotides (morpholinos) in zebrafish has led to a range of developmental defects. This study characterised zebrafish embryos homozygous for atoh8sa1465, a loss-of-function allele of atoh8, in order to provide genetic evidence for the developmental role of Atoh8 in this species. Embryos homozygous for atoh8sa1465 present normal body morphology, swimbladder inflation, and heart looping, and survive to adulthood. These embryos do not develop pericardial oedema by 72 hpf and are not sensitised to the loss of Fog1 protein, suggesting that this previously described abnormality is not a specific phenotype. Vascular patterning and primitive haematopoiesis are unaffected in atoh8sa1465/sa1465 mutant embryos. Together, the data suggest that Atoh8 is dispensible for zebrafish development under standard laboratory conditions. PMID:28182631

  20. Deletion of Pr130 Interrupts Cardiac Development in Zebrafish

    PubMed Central

    Yang, Jie; Li, Zuhua; Gan, Xuedong; Zhai, Gang; Gao, Jiajia; Xiong, Chenling; Qiu, Xueping; Wang, Xuebin; Yin, Zhan; Zheng, Fang

    2016-01-01

    Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A), a regulatory subunit of protein phosphatase 2A (PP2A), is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR), plays an important role in the excitation-contraction (EC) coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system. Pr130-knockout zebrafish exhibited cardiac looping defects and decreased cardiac function (decreased fractional area and fractional shortening). Hematoxylin and eosin (H&E) staining demonstrated reduced cardiomyocytes. Subsequent transmission electron microscopy revealed that the bright and dark bands were narrowed and blurred, the Z- and M-lines were fogged, and the gaps between longitudinal myocardial fibers were increased. Additionally, increased apoptosis was observed in cardiomyocyte in pr130-knockout zebrafish compared to wild-type (WT). Taken together, our results suggest that pr130 is required for normal myocardium formation and efficient cardiac contractile function. PMID:27845735

  1. A crystal-clear zebrafish for in vivo imaging

    PubMed Central

    Antinucci, Paride; Hindges, Robert

    2016-01-01

    The larval zebrafish (Danio rerio) is an excellent vertebrate model for in vivo imaging of biological phenomena at subcellular, cellular and systems levels. However, the optical accessibility of highly pigmented tissues, like the eyes, is limited even in this animal model. Typical strategies to improve the transparency of zebrafish larvae require the use of either highly toxic chemical compounds (e.g. 1-phenyl-2-thiourea, PTU) or pigmentation mutant strains (e.g. casper mutant). To date none of these strategies produce normally behaving larvae that are transparent in both the body and the eyes. Here we present crystal, an optically clear zebrafish mutant obtained by combining different viable mutations affecting skin pigmentation. Compared to the previously described combinatorial mutant casper, the crystal mutant lacks pigmentation also in the retinal pigment epithelium, therefore enabling optical access to the eyes. Unlike PTU-treated animals, crystal larvae are able to perform visually guided behaviours, such as the optomotor response, as efficiently as wild type larvae. To validate the in vivo application of crystal larvae, we performed whole-brain light-sheet imaging and two-photon calcium imaging of neural activity in the retina. In conclusion, this novel combinatorial pigmentation mutant represents an ideal vertebrate tool for completely unobstructed structural and functional in vivo investigations of biological processes, particularly when imaging tissues inside or between the eyes. PMID:27381182

  2. Zebrafish Behavior: Opportunities and Challenges.

    PubMed

    Orger, Michael B; de Polavieja, Gonzalo G

    2017-04-03

    A great challenge in neuroscience is understanding how activity in the brain gives rise to behavior. The zebrafish is an ideal vertebrate model to address this challenge, thanks to the capacity, at the larval stage, for precise behavioral measurements, genetic manipulations, and recording and manipulation of neural activity noninvasively and at single-neuron resolution throughout the whole brain. These techniques are being further developed for application in freely moving animals and juvenile stages to study more complex behaviors including learning, decision making, and social interactions. We review some of the approaches that have been used to study the behavior of zebrafish and point to opportunities and challenges that lie ahead. Expected final online publication date for the Annual Review of Neuroscience Volume 40 is July 8, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  3. DNA repair capacity of zebrafish.

    PubMed

    Sussman, Raquel

    2007-08-14

    Damage to the genome is unavoidable in living creatures, because of sunlight exposure as well as environmental chemicals present in food and drinking water. There is a need to monitor and purify the drinking water; therefore, several methods of detection have been developed. A very promising model system for this purpose is the zebrafish (Danio rerio), which is endowed with special qualities for detecting external as well as internal abnormalities. Grossman and Wei's assay [Grossman L, Wei Q (1995) Clin Chem 12:1854-1863], which measures the expression level of a nonreplicating recombinant plasmid DNA containing a UV-damaged luciferase reporter gene, shows that zebrafish can repair chromosomal lesions to a much greater extent than the human population. This vertebrate model is still very promising after possible down-regulation of the DNA repair enzymes.

  4. Diet affects spawning in zebrafish.

    PubMed

    Markovich, Michelle L; Rizzuto, Noel V; Brown, Paul B

    2007-01-01

    Seven-month-old zebrafish (Danio rerio) were fed four different diets to test the hypothesis that diet affects spawning success and resulting characteristics of eggs and offspring. The diets were: the recommended feeding regime for zebrafish (a mixture of Artemia, flake feed, and liver paste); Artemia; a flake feed; and a commercially available trout diet. The number of eggs laid and average egg diameter were significantly different as functions of male, female, and individual matings. Fish fed the flake diet produced significantly fewer eggs (mean, 116) than fish fed all other diets (means, 166-187). However, the percent hatch of eggs from fish fed the flake diet (62.5%) was significantly higher than from fish fed the trout diet (19.5%). The percentages of hatched eggs from fish fed the control diet (36.2%) or Artemia (35.6%) were not significantly different from each other or from fish fed the other two diets. Wet weight and diameter of eggs were not significantly affected by diet. Larval length was significantly higher from parents fed the flake diet (14.5 mm) compared to larvae from parents fed Artemia (13.7 mm). Length of larvae from fish fed the control or trout diets was intermediate and not significantly different from fish fed the flake diet or Artemia. Larval weight was not significantly affected by dietary treatment, but offspring from fish fed the flake diet were heavier than larvae from adults fed any of the other diets. Feeding adult zebrafish the flake diet alone resulted in more viable offspring and larger larvae and is a simpler feeding regime than the current recommendation. The authors recommend feeding adult zebrafish flake diets to satiation three times daily for maximum production of viable offspring.

  5. Zebrafish genomics comes of age.

    PubMed

    Tan, Haihan; Zsigmond, Aron

    2013-09-01

    The ZF-HEALTH/EuFishBiomed workshop on "Genomics and High-throughput Sequencing Technologies with the Zebrafish Model" took place in December 2012 in Cambridge, United Kingdom. The organisers, Fiona Wardle and Ferenc Müller, brought together developmental biologists, geneticists, and bioinformaticians from Europe and the rest of the world to share findings and insights about the latest genomic capabilities and applications in this popular model organism.

  6. Mapping the development of cerebellar Purkinje cells in zebrafish.

    PubMed

    Hamling, Kyla R; Tobias, Zachary J C; Weissman, Tamily A

    2015-11-01

    The cells that comprise the cerebellum perform a complex integration of neural inputs to influence motor control and coordination. The functioning of this circuit depends upon Purkinje cells and other cerebellar neurons forming in the precise place and time during development. Zebrafish provide a useful platform for modeling disease and studying gene function, thus a quantitative metric of normal zebrafish cerebellar development is key for understanding how gene mutations affect the cerebellum. To begin to quantitatively measure cerebellar development in zebrafish, we have characterized the spatial and temporal patterning of Purkinje cells during the first 2 weeks of development. Differentiated Purkinje cells first emerged by 2.8 days post fertilization and were spatially patterned into separate dorsomedial and ventrolateral clusters that merged at around 4 days. Quantification of the Purkinje cell layer revealed that there was a logarithmic increase in both Purkinje cell number as well as overall volume during the first 2 weeks, while the entire region curved forward in an anterior, then ventral direction. Purkinje cell dendrites were positioned next to parallel fibers as early as 3.3 days, and Purkinje cell diameter decreased significantly from 3.3 to 14 days, possibly due to cytoplasmic reappropriation into maturing dendritic arbors. A nearest neighbor analysis showed that Purkinje cells moved slightly apart from each other from 3 to 14 days, perhaps spreading as the organized monolayer forms. This study establishes a quantitative spatiotemporal map of Purkinje cell development in zebrafish that provides an important metric for studies of cerebellar development and disease. © 2015 Wiley Periodicals, Inc.

  7. Developmental effects of simulated microgravity on zebrafish, (Danio rerio)

    NASA Astrophysics Data System (ADS)

    Stoyek, Matthew; Edsall, Sara; Franz-Odendaal, Tamara; Smith, Frank; Croll, Roger

    Zebrafish are widely used model vertebrates in research and recently this species has been used to study the effects of microgravity on fundamental biological processes. In this study we used a NASA-designed rotating wall vessel (RWV) to investigate the effects of simulated microgravity (SMG) on zebrafish development up to 14 days post fertilization (dpf). At developmental stages beyond the 3-4 somite stage we found SMG-exposed embryos reached key developmental stag-ing points more rapidly than fish raised within a non-rotating vessel. By the 21 somite stage, both groups were again synchronized in their developmental staging. However, SMG-exposed embryos eventually exhibited a delay in hatching time compared to controls. Otolith and to-tal body size were observed to be greater in larvae raised in SMG. In addition, pigmentation patterns in SMG exposed fish differed, with larger and differentially aggregated melanocytes . Heart development was slowed in SMG exposed fish, but no change in nervous system de-velopment was detected. Ongoing research will focus on differences in heart and respiration rates. Finally, by developing a method to extend the duration of SMG exposure, we found the swimming behaviour of SMG-exposed animals was altered with time in the RWV. Initially SMG-exposed animals swam in the direction of RWV rotation (5-9dpf) but older (9+dpf) fish swam against rotation and demonstrated righting behaviour with each rotation. These results suggest that vestibular reflexes may develop normally and be maintained in animals exposed to SMG. Together, our data provide insights into how zebrafish may develop when flown in space, permitting better formulation of experiments to test mechanisms by which microgravity may affect ontogeny of this model organism. Keywords: microgravity, zebrafish, growth, development

  8. Inflammatory diseases modelling in zebrafish

    PubMed Central

    Morales Fénero, Camila Idelí; Colombo Flores, Alicia Angelina; Câmara, Niels Olsen Saraiva

    2016-01-01

    The ingest of diets with high content of fats and carbohydrates, low or no physical exercise and a stressful routine are part of the everyday lifestyle of most people in the western world. These conditions are triggers for different diseases with complex interactions between the host genetics, the metabolism, the immune system and the microbiota, including inflammatory bowel diseases (IBD), obesity and diabetes. The incidence of these disorders is growing worldwide; therefore, new strategies for its study are needed. Nowadays, the majority of researches are in use of murine models for understand the genetics, physiopathology and interaction between cells and signaling pathways to find therapeutic solutions to these diseases. The zebrafish, a little tropical water fish, shares 70% of our genes and conserves anatomic and physiological characteristics, as well as metabolical pathways, with mammals, and is rising as a new complementary model for the study of metabolic and inflammatory diseases. Its high fecundity, fast development, transparency, versatility and low cost of maintenance makes the zebrafish an interesting option for new researches. In this review, we offer a discussion of the existing genetic and induced zebrafish models of two important Western diseases that have a strong inflammatory component, the IBD and the obesity. PMID:26929916

  9. Molecular analysis and heavy metal detoxification of ABCC1/MRP1 in zebrafish.

    PubMed

    Long, Yong; Li, Qing; Cui, Zongbin

    2011-03-01

    ABCC1/MRP1 belongs to the ATP-binding cassette superfamily and its elevated expression is closely associated with the multidrug resistance of various tumor cells. In normal tissues, ABCC1 confers resistance to a wide variety of xenobiotics and toxicants, demonstrating its important roles in tissue defense. Here, we report the cloning and functional characterization of abcc1 gene in zebrafish. This gene is localized on zebrafish chromosome 3 and contains a 4,557 bp open-reading frame. The deduced polypeptide is composed of 1,518 amino acids, which shares 70% identity with human ABCC1. Phylogenetic analysis revealed that ABCC1 proteins from thirteen vertebrate species are highly conserved during evolution. Transcriptional expression of zebrafish abcc1 gene in developing embryos was examined by whole-mount in situ hybridization and real-time PCR. Transcripts of zebrafish abcc1 gene were detectable in four-cell stage embryos, indicating that this gene is maternally expressed. ABCC1 mRNAs were ubiquitously distributed in embryos before 12 h post-fertilization (hpf) and mainly localized in eyes and brain from 24 to 72 hpf, and in gills from 96 to 120 hpf. In addition, zebrafish abcc1 gene was highly expressed in 1-hpf embryos and detected in all adult tissues examined, with highest expression in testis and lowest in heart and liver. Exposure of ZF4 cells and embryos to CdCl(2)·2.5H(2)O, HgCl(2), Pb(NO(3))(2), or Na(3)AsO(4)·12H(2)O significantly induced transcriptional expression of abcc1 gene. Furthermore, overexpression of abcc1 improved the survival rates of embryos exposed to Cd, Hg or As, while overexpression of a abcc1 mutant (ABCC1-G1420D) sensitized zebrafish embryos to toxic metals. These data indicate that zebrafish ABCC1 has crucial roles in heavy metals detoxification.

  10. Disruption of LRRK2 Does Not Cause Specific Loss of Dopaminergic Neurons in Zebrafish

    PubMed Central

    Ren, Guiqi; Xin, Shengchang; Li, Song; Zhong, Hanbing; Lin, Shuo

    2011-01-01

    Mutations in LRRK2 are genetically linked to Parkinson's disease (PD) but its normal biological function is largely unknown. Sheng et al. recently reported that deletion of the WD40 domain of LRRK2 in zebrafish specifically causes PD-like loss of neurons and behavior defect. However, our similar early study and recent confirming experiments using the same reagents reported by Sheng et al. failed to reproduce the phenotype of the loss of dopaminergic neurons, although the mRNA of LRRK2 was molecularly disrupted. Our study suggests that function of LRRK2 and its usefulness to generate zebrafish PD model needs further evaluation. PMID:21698186

  11. Featured organism: Danio rerio, the zebrafish.

    PubMed

    Wixon, J

    2000-09-30

    The zebrafish has long been a favourite model for the study of vertebrate development. Here we provide an overview of the current state of knowledge and resources for the study of this fish, with comments on the future direction of zebrafish genomics from Professor Mark Fishman and Dr Stephen Wilson.

  12. Zebrafish as a model for human osteosarcoma.

    PubMed

    Mohseny, A B; Hogendoorn, P C W

    2014-01-01

    For various reasons involving biological comparativeness, expansive technological possibilities, accelerated experimental speed, and competitive costs, zebrafish has become a comprehensive model for cancer research. Hence, zebrafish embryos and full-grown fish have been instrumental for studies of leukemia, melanoma, pancreatic cancer, bone tumors, and other malignancies. Although because of its similarities to human osteogenesis zebrafish appears to be an appealing model to investigate osteosarcoma, only a few osteosarcoma specific studies have been accomplished yet. Here, we review interesting related and unrelated reports of which the findings might be extrapolated to osteosarcoma. More importantly, rational but yet unexplored applications of zebrafish are debated to expand the window of opportunities for future establishment of osteosarcoma models. Accordingly technological advances of zebrafish based cancer research, such as robotic high-throughput multicolor injection systems and advanced imaging methods are discussed. Furthermore, various use of zebrafish embryos for screening drug regimens by combinations of chemotherapy, novel drug deliverers, and immune system modulators are suggested. Concerning the etiology, the high degree of genetic similarity between zebrafish and human cancers indicates that affected regions are evolutionarily conserved. Therefore, zebrafish as a swift model system that allows for the investigation of multiple candidate gene-defects is presented.

  13. A Comparative Map of the Zebrafish Genome

    PubMed Central

    Woods, Ian G.; Kelly, Peter D.; Chu, Felicia; Ngo-Hazelett, Phuong; Yan, Yi-Lin; Huang, Hui; Postlethwait, John H.; Talbot, William S.

    2000-01-01

    Zebrafish mutations define the functions of hundreds of essential genes in the vertebrate genome. To accelerate the molecular analysis of zebrafish mutations and to facilitate comparisons among the genomes of zebrafish and other vertebrates, we used a homozygous diploid meiotic mapping panel to localize polymorphisms in 691 previously unmapped genes and expressed sequence tags (ESTs). Together with earlier efforts, this work raises the total number of markers scored in the mapping panel to 2119, including 1503 genes and ESTs and 616 previously characterized simple-sequence length polymorphisms. Sequence analysis of zebrafish genes mapped in this study and in prior work identified putative human orthologs for 804 zebrafish genes and ESTs. Map comparisons revealed 139 new conserved syntenies, in which two or more genes are on the same chromosome in zebrafish and human. Although some conserved syntenies are quite large, there were changes in gene order within conserved groups, apparently reflecting the relatively frequent occurrence of inversions and other intrachromosomal rearrangements since the divergence of teleost and tetrapod ancestors. Comparative mapping also shows that there is not a one-to-one correspondence between zebrafish and human chromosomes. Mapping of duplicate gene pairs identified segments of 20 linkage groups that may have arisen during a genome duplication that occurred early in the evolution of teleosts after the divergence of teleost and mammalian ancestors. This comparative map will accelerate the molecular analysis of zebrafish mutations and enhance the understanding of the evolution of the vertebrate genome. PMID:11116086

  14. A Novel Method for Rearing Zebrafish by Using Freshwater Rotifers (Brachionus calyciflorus).

    PubMed

    Aoyama, Yuta; Moriya, Natsumi; Tanaka, Shingo; Taniguchi, Tomoko; Hosokawa, Hiroshi; Maegawa, Shingo

    2015-08-01

    The zebrafish (Danio rerio) has become a powerful model organism for studying developmental processes and genetic diseases. However, there remain several problems in previous rearing methods. In this study, we demonstrate a novel method for rearing zebrafish larvae by using a new first food, freshwater rotifers (Brachionus calyciflorus). Feeding experiments indicated that freshwater rotifers are suitable as the first food for newly hatched larval fish. In addition, we revisited and improved a feeding schedule from 5 to 40 days postfertilization. Our feeding method using freshwater rotifers accelerated larval growth. At 49 dpf, one pair out of 10 pairs successfully produced six fertilized eggs. At 56, 63, and 71 dpf, 6 out of the 10 pairs constantly produced normal embryos. Our method will improve the husbandry of the zebrafish.

  15. A Novel Method for Rearing Zebrafish by Using Freshwater Rotifers (Brachionus calyciflorus)

    PubMed Central

    Aoyama, Yuta; Moriya, Natsumi; Tanaka, Shingo; Taniguchi, Tomoko; Hosokawa, Hiroshi

    2015-01-01

    Abstract The zebrafish (Danio rerio) has become a powerful model organism for studying developmental processes and genetic diseases. However, there remain several problems in previous rearing methods. In this study, we demonstrate a novel method for rearing zebrafish larvae by using a new first food, freshwater rotifers (Brachionus calyciflorus). Feeding experiments indicated that freshwater rotifers are suitable as the first food for newly hatched larval fish. In addition, we revisited and improved a feeding schedule from 5 to 40 days postfertilization. Our feeding method using freshwater rotifers accelerated larval growth. At 49 dpf, one pair out of 10 pairs successfully produced six fertilized eggs. At 56, 63, and 71 dpf, 6 out of the 10 pairs constantly produced normal embryos. Our method will improve the husbandry of the zebrafish. PMID:25938499

  16. Novel use of zebrafish as a vertebrate model to screen radiation protectors and sensitizers

    SciTech Connect

    McAleer, Mary Frances . E-mail: adam.dicker@mail.tju.edu; Davidson, Christian; Davidson, William Robert; Yentzer, Brad; Farber, Steven A.; Rodeck, Ulrich; Dicker, Adam P.

    2005-01-01

    Purpose: Zebrafish (Danio rerio) embryos provide a unique vertebrate model to screen therapeutic agents easily and rapidly because of their relatively close genetic relationship to humans, ready abundance and accessibility, short embryonal development, and optical clarity. To validate zebrafish embryos as a screen for radiation modifiers, we evaluated the effects of ionizing radiation in combination with a known radioprotector (free radical scavenger Amifostine) or radiosensitizing agent (tyrosine kinase inhibitor AG1478). Methods and materials: Viable zebrafish embryos were exposed to 0-10 Gy single-fraction 250 kVp X-rays with or without either Amifostine (0-4 mM) or AG1478 (0-10 {mu}M) at defined developmental stages from 1-24 h postfertilization (hpf). Embryos were examined for morphologic abnormalities and viability until 144 hpf. Results: Radiation alone produced a time- and dose-dependent perturbation of normal embryonic development and survival with maximal sensitivity at doses {>=}4 Gy delivered before 4 hpf. Amifostine markedly attenuated this effect, whereas AG1478 enhanced teratogenicity and lethality, particularly at therapeutically relevant (2-6 Gy) radiation doses. Conclusions: Collectively, these data validate the use of zebrafish as a vertebrate model to assess the effect of radiation alone or with radiation response modulators. Zebrafish embryos may thus provide a rapid, facile system to screen novel agents ultimately intended for human use in the context of therapeutic or accidental radiation exposure.

  17. The zebrafish eye-a paradigm for investigating human ocular genetics.

    PubMed

    Richardson, R; Tracey-White, D; Webster, A; Moosajee, M

    2017-01-01

    Although human epidemiological and genetic studies are essential to elucidate the aetiology of normal and aberrant ocular development, animal models have provided us with an understanding of the pathogenesis of multiple developmental ocular malformations. Zebrafish eye development displays in depth molecular complexity and stringent spatiotemporal regulation that incorporates developmental contributions of the surface ectoderm, neuroectoderm and head mesenchyme, similar to that seen in humans. For this reason, and due to its genetic tractability, external fertilisation, and early optical clarity, the zebrafish has become an invaluable vertebrate system to investigate human ocular development and disease. Recently, zebrafish have been at the leading edge of preclinical therapy development, with their amenability to genetic manipulation facilitating the generation of robust ocular disease models required for large-scale genetic and drug screening programmes. This review presents an overview of human and zebrafish ocular development, genetic methodologies employed for zebrafish mutagenesis, relevant models of ocular disease, and finally therapeutic approaches, which may have translational leads in the future.

  18. Alcohol-Induced Morphological Deficits in the Development of Octavolateral Organs of the Zebrafish (Danio rerio)

    PubMed Central

    Zamora, Lilliann Y.

    2013-01-01

    Abstract Prenatal alcohol exposure is known to have many profound detrimental effects on human fetal development (fetal alcohol spectrum disorders), which may manifest as lifelong disabilities. However, how alcohol affects the auditory/vestibular system is still largely unknown. This is the first study to investigate morphological effects of alcohol on the developing octavolateral system (the inner ear and lateral line) using the zebrafish, Danio rerio. Zebrafish embryos of 2 hours post fertilization (hpf) were treated in 2% alcohol for 48 hours and screened at 72 hpf for morphological defects of the inner ear and lateral line. Octavolateral organs from both alcohol-treated and control zebrafish were examined using light, confocal, and scanning electron microscopy. We observed several otolith phenotypes for alcohol-treated zebrafish including zero, one, two abnormal, two normal, and multiple otoliths. Results of this study show that alcohol treatment during early development impairs the inner ear (smaller ear, abnormal otoliths, and fewer sensory hair cells) and the lateral line (smaller neuromasts, fewer neuromasts and hair cells per neuromast, and shorter kinocilia of hair cells). Early embryonic alcohol exposure may also result in defects in hearing, balance, and hydrodynamic function of zebrafish. PMID:23461415

  19. Alcohol-induced morphological deficits in the development of octavolateral organs of the zebrafish (Danio rerio).

    PubMed

    Zamora, Lilliann Y; Lu, Zhongmin

    2013-03-01

    Prenatal alcohol exposure is known to have many profound detrimental effects on human fetal development (fetal alcohol spectrum disorders), which may manifest as lifelong disabilities. However, how alcohol affects the auditory/vestibular system is still largely unknown. This is the first study to investigate morphological effects of alcohol on the developing octavolateral system (the inner ear and lateral line) using the zebrafish, Danio rerio. Zebrafish embryos of 2 hours post fertilization (hpf) were treated in 2% alcohol for 48 hours and screened at 72 hpf for morphological defects of the inner ear and lateral line. Octavolateral organs from both alcohol-treated and control zebrafish were examined using light, confocal, and scanning electron microscopy. We observed several otolith phenotypes for alcohol-treated zebrafish including zero, one, two abnormal, two normal, and multiple otoliths. Results of this study show that alcohol treatment during early development impairs the inner ear (smaller ear, abnormal otoliths, and fewer sensory hair cells) and the lateral line (smaller neuromasts, fewer neuromasts and hair cells per neuromast, and shorter kinocilia of hair cells). Early embryonic alcohol exposure may also result in defects in hearing, balance, and hydrodynamic function of zebrafish.

  20. Mesoderm is required for coordinated cell movements within zebrafish neural plate in vivo

    PubMed Central

    2014-01-01

    Background Morphogenesis of the zebrafish neural tube requires the coordinated movement of many cells in both time and space. A good example of this is the movement of the cells in the zebrafish neural plate as they converge towards the dorsal midline before internalizing to form a neural keel. How these cells are regulated to ensure that they move together as a coherent tissue is unknown. Previous work in other systems has suggested that the underlying mesoderm may play a role in this process but this has not been shown directly in vivo. Results Here we analyze the roles of subjacent mesoderm in the coordination of neural cell movements during convergence of the zebrafish neural plate and neural keel formation. Live imaging demonstrates that the normal highly coordinated movements of neural plate cells are lost in the absence of underlying mesoderm and the movements of internalization and neural tube formation are severely disrupted. Despite this, neuroepithelial polarity develops in the abnormal neural primordium but the resulting tissue architecture is very disorganized. Conclusions We show that the movements of cells in the zebrafish neural plate are highly coordinated during the convergence and internalization movements of neurulation. Our results demonstrate that the underlying mesoderm is required for these coordinated cell movements in the zebrafish neural plate in vivo. PMID:24755297

  1. Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Lei, Yong; Xiao, Qi; Huang, Shan; Xu, Wansu; Zhang, Zhe; He, Zhike; Liu, Yi; Deng, Fengjiao

    2011-12-01

    Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.

  2. Behavioral and biochemical adjustments of the zebrafish Danio rerio exposed to the β-blocker propranolol.

    PubMed

    Mitchell, Kimberly M; Moon, Thomas W

    2016-09-01

    Propranolol (PROP) is a β-blocker prescribed mainly to treat human cardiovascular diseases and as a result of its wide usage and persistence, it is reported in aquatic environments. This study examined whether PROP alters developmental patterns and catecholamine (CA)-regulated processes in the zebrafish (Danio rerio) and if exposure during early life alters the stress response and behaviors of adults. The calculated 48h larva LC50 was 21.6mg/L, well above reported environmental levels (0.01-0.59μg/L). Stressed and PROP-exposed adult zebrafish had reduced testosterone and estradiol levels and exhibited behaviors indicating less anxiety than control fish. Furthermore, adults previously PROP-exposed as embryos/larvae had decreased growth in terms of body length and mass. Finally, these adults showed increased cholesterol and a dose-dependent decrease in testosterone levels compared with unexposed zebrafish. Thus PROP-exposure of zebrafish embryos/larvae alters developmental patterns and CA-regulated processes that may affect normal behaviors and responses to stressors, and at least some of these changes persist in the adult zebrafish. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Upregulation of leukemia inhibitory factor (LIF) during the early stage of optic nerve regeneration in zebrafish.

    PubMed

    Ogai, Kazuhiro; Kuwana, Ayaka; Hisano, Suguru; Nagashima, Mikiko; Koriyama, Yoshiki; Sugitani, Kayo; Mawatari, Kazuhiro; Nakashima, Hiroshi; Kato, Satoru

    2014-01-01

    Fish retinal ganglion cells (RGCs) can regenerate their axons after optic nerve injury, whereas mammalian RGCs normally fail to do so. Interleukin 6 (IL-6)-type cytokines are involved in cell differentiation, proliferation, survival, and axon regrowth; thus, they may play a role in the regeneration of zebrafish RGCs after injury. In this study, we assessed the expression of IL-6-type cytokines and found that one of them, leukemia inhibitory factor (LIF), is upregulated in zebrafish RGCs at 3 days post-injury (dpi). We then demonstrated the activation of signal transducer and activator of transcription 3 (STAT3), a downstream target of LIF, at 3-5 dpi. To determine the function of LIF, we performed a LIF knockdown experiment using LIF-specific antisense morpholino oligonucleotides (LIF MOs). LIF MOs, which were introduced into zebrafish RGCs via a severed optic nerve, reduced the expression of LIF and abrogated the activation of STAT3 in RGCs after injury. These results suggest that upregulated LIF drives Janus kinase (Jak)/STAT3 signaling in zebrafish RGCs after nerve injury. In addition, the LIF knockdown impaired axon sprouting in retinal explant culture in vitro; reduced the expression of a regeneration-associated molecule, growth-associated protein 43 (GAP-43); and delayed functional recovery after optic nerve injury in vivo. In this study, we comprehensively demonstrate the beneficial role of LIF in optic nerve regeneration and functional recovery in adult zebrafish.

  4. Lrrc10 is required for early heart development and function in zebrafish

    PubMed Central

    Kim, Ki-Hyun; Antkiewicz, Dagmara S.; Yan, Long; Eliceiri, Kevin W.; Heideman, Warren; Peterson, Richard E.; Lee, Youngsook

    2007-01-01

    Leucine-rich Repeat Containing protein 10 (LRRC10) has recently been identified as a cardiac-specific factor in mice. However, the function of this factor remains to be elucidated. In this study, we investigated the developmental roles of Lrrc10 using zebrafish as an animal model. Knockdown of Lrrc10 in zebrafish embryos (morphants) using morpholinos caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and embryonic lethality between day 6 and 7 post fertilization. The Lrrc10 morphants exhibited cardiac functional defects as evidenced by a decrease in ejection fraction and cardiac output. Further investigations into the underlying mechanisms of the cardiac defects revealed that the number of cardiomyocyte was reduced in the morphants. Expression of two cardiac genes was deregulated in the morphants including an increase in atrial natriuretic factor, a hallmark for cardiac hypertrophy and failure, and a decrease in cardiac myosin light chain 2, an essential protein for cardiac contractility in zebrafish. Moreover, a reduced fluorescence intensity from NADH in the morphant heart was observed in live zebrafish embryos as compared to control. Taken together, the present study demonstrates that Lrrc10 is necessary for normal cardiac development and cardiac function in zebrafish embryos, which will enhance our understanding of congenital heart defects and heart disease. PMID:17601532

  5. Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1) Mutant Zebrafish

    PubMed Central

    Grone, Brian P.; Marchese, Maria; Hamling, Kyla R.; Kumar, Maneesh G.; Krasniak, Christopher S.; Sicca, Federico; Santorelli, Filippo M.; Patel, Manisha; Baraban, Scott C.

    2016-01-01

    Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1), are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b) have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing “dark-flash” visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations. PMID:26963117

  6. Optimized cell transplantation using adult rag2 mutant zebrafish

    PubMed Central

    Tang, Qin; Abdelfattah, Nouran S.; Blackburn, Jessica S.; Moore, John C.; Martinez, Sarah A.; Moore, Finola E.; Lobbardi, Riadh; Tenente, Inês M.; Ignatius, Myron S.; Berman, Jason N.; Liwski, Robert S.; Houvras, Yariv; Langenau, David M.

    2014-01-01

    Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer. PMID:25042784

  7. Zebrafish: modeling for herpes simplex virus infections.

    PubMed

    Antoine, Thessicar Evadney; Jones, Kevin S; Dale, Rodney M; Shukla, Deepak; Tiwari, Vaibhav

    2014-02-01

    For many years, zebrafish have been the prototypical model for studies in developmental biology. In recent years, zebrafish has emerged as a powerful model system to study infectious diseases, including viral infections. Experiments conducted with herpes simplex virus type-1 in adult zebrafish or in embryo models are encouraging as they establish proof of concept with viral-host tropism and possible screening of antiviral compounds. In addition, the presence of human homologs of viral entry receptors in zebrafish such as 3-O sulfated heparan sulfate, nectins, and tumor necrosis factor receptor superfamily member 14-like receptor bring strong rationale for virologists to test their in vivo significance in viral entry in a zebrafish model and compare the structure-function basis of virus zebrafish receptor interaction for viral entry. On the other end, a zebrafish model is already being used for studying inflammation and angiogenesis, with or without genetic manipulations, and therefore can be exploited to study viral infection-associated pathologies. The major advantage with zebrafish is low cost, easy breeding and maintenance, rapid lifecycle, and a transparent nature, which allows visualizing dissemination of fluorescently labeled virus infection in real time either at a localized region or the whole body. Further, the availability of multiple transgenic lines that express fluorescently tagged immune cells for in vivo imaging of virus infected animals is extremely attractive. In addition, a fully developed immune system and potential for receptor-specific knockouts further advocate the use of zebrafish as a new tool to study viral infections. In this review, we focus on expanding the potential of zebrafish model system in understanding human infectious diseases and future benefits.

  8. Zebrafish: Modeling for Herpes Simplex Virus Infections

    PubMed Central

    Antoine, Thessicar Evadney; Jones, Kevin S.; Dale, Rodney M.; Shukla, Deepak

    2014-01-01

    Abstract For many years, zebrafish have been the prototypical model for studies in developmental biology. In recent years, zebrafish has emerged as a powerful model system to study infectious diseases, including viral infections. Experiments conducted with herpes simplex virus type-1 in adult zebrafish or in embryo models are encouraging as they establish proof of concept with viral-host tropism and possible screening of antiviral compounds. In addition, the presence of human homologs of viral entry receptors in zebrafish such as 3-O sulfated heparan sulfate, nectins, and tumor necrosis factor receptor superfamily member 14-like receptor bring strong rationale for virologists to test their in vivo significance in viral entry in a zebrafish model and compare the structure–function basis of virus zebrafish receptor interaction for viral entry. On the other end, a zebrafish model is already being used for studying inflammation and angiogenesis, with or without genetic manipulations, and therefore can be exploited to study viral infection-associated pathologies. The major advantage with zebrafish is low cost, easy breeding and maintenance, rapid lifecycle, and a transparent nature, which allows visualizing dissemination of fluorescently labeled virus infection in real time either at a localized region or the whole body. Further, the availability of multiple transgenic lines that express fluorescently tagged immune cells for in vivo imaging of virus infected animals is extremely attractive. In addition, a fully developed immune system and potential for receptor-specific knockouts further advocate the use of zebrafish as a new tool to study viral infections. In this review, we focus on expanding the potential of zebrafish model system in understanding human infectious diseases and future benefits. PMID:24266790

  9. Zebrafish Endzone Regulates Neural Crest-Derived Chromatophore Differentiation and Morphology

    PubMed Central

    Arduini, Brigitte L.; Gallagher, Glen R.; Henion, Paul D.

    2008-01-01

    The development of neural crest-derived pigment cells has been studied extensively as a model for cellular differentiation, disease and environmental adaptation. Neural crest-derived chromatophores in the zebrafish (Danio rerio) consist of three types: melanophores, xanthophores and iridiphores. We have identified the zebrafish mutant endzone (enz), that was isolated in a screen for mutants with neural crest development phenotypes, based on an abnormal melanophore pattern. We have found that although wild-type numbers of chromatophore precursors are generated in the first day of development and migrate normally in enz mutants, the numbers of all three chromatophore cell types that ultimately develop are reduced. Further, differentiated melanophores and xanthophores subsequently lose dendricity, and iridiphores are reduced in size. We demonstrate that enz function is required cell autonomously by melanophores and that the enz locus is located on chromosome 7. In addition, zebrafish enz appears to selectively regulate chromatophore development within the neural crest lineage since all other major derivatives develop normally. Our results suggest that enz is required relatively late in the development of all three embryonic chromatophore types and is normally necessary for terminal differentiation and the maintenance of cell size and morphology. Thus, although developmental regulation of different chromatophore sublineages in zebrafish is in part genetically distinct, enz provides an example of a common regulator of neural crest-derived chromatophore differentiation and morphology. PMID:18665240

  10. Mmp23b promotes liver development and hepatocyte proliferation through the TNF pathway in zebrafish

    PubMed Central

    Qi, Fei; Song, Jianbo; Yang, Hanshuo; Gao, Wei; Liu, Ning-ai; Zhang, Bo; Lin, Shuo

    2012-01-01

    The matrix metalloproteinase (MMP) family of proteins degrades extracellular matrix (ECM) components as well as processes cytokines and growth factors. MMPs are involved in regulating ECM homeostasis in both normal physiology and disease pathophysiology. Here, we report the critical roles of mmp23b in normal zebrafish liver development. Mmp23b was initially identified as a gene linked to the genomic locus of an enhancer trap transgenic zebrafish line in which GFP expression was restricted to the developing liver. Follow-up analysis of mmp23b mRNA expression confirmed its liver-specific expression pattern. Morpholino (MO) knockdown of mmp23b resulted in defective hepatocyte proliferation, causing a reduction in liver size while maintaining relatively normal pancreas and gut development. Genetically, we showed that mmp23b functions through the tumor necrosis factor (TNF) signaling pathway. Antisense knockdown of tnfa or tnfb in zebrafish caused similar reductions of liver size whereas overexpression of tnfa or tnfb rescued liver defects in mmp23b morphants but not vice versa. Biochemically, MMP23B, the human ortholog of Mmp23b, directly interacts with TNF and mediates its release from the cell membrane in a cell culture system. Since mmp23b/MMP23B is highly conserved, our findings in zebrafish warrant further investigation of its role in regulating liver development in mammals. PMID:21064033

  11. Tenascin-C expression in the trunk of wild-type, cyclops and floating head zebrafish embryos.

    PubMed

    Tongiorgi, E

    1999-01-01

    The function and the regulation of the expression of the extracellular matrix molecule tenascin-C during embryonic development are still unclear. In the present study, the expression of tenascin-C was analyzed in the trunk of zebrafish at the end of the first embryonic day. An antiserum raised against a zebrafish tenascin-C (TN-C) fusion protein reacted with 220 (doublet), 200, and 160 KD peptides. In situ hybridization showed that in the zebrafish wild-type embryo, tn-c mRNA was expressed by somites, neural crest cells, roof plate, notochord, hypochord, and tail fin bud. Thus, the expression of tn-c mRNA is an excellent marker for the differentiation of most zebrafish trunk structures. Immunolabelling with the anti-TN-C antibody was detected in the migratory pathway of neural crest cells and in the intersomitic furrows. In situ hybridization analysis of the zebrafish cyclops mutants, lacking the midline floor plate cells, showed normal expression of tn-c mRNA in all trunk structures. Analysis of the floating-head mutant, lacking the notochord, showed that tn-c mRNA expression in neural crest cells, roof plate, and tail fin bud is normal, but it is defective in the somites. By showing that the notochord, but not the floor plate, cells are required for normal tn-c expression in the trunk, this work provides new information on the role played by the embryonic axial structures in the regulation of the expression of tn-c during the development of zebrafish and allows new conclusions about somite patterning in the cyclops and floating-head zebrafish mutants.

  12. Investigation of the Role of Stress in Inflammatory Bowel Disease Using Zebrafish as an Experimental Model

    DTIC Science & Technology

    2012-08-01

    pathogenesis. For instance, while stress has for many years been implicated in symptom precipitation, the role of the normal gut flora (microbiome) has only...expressed as arbitrary mRNA units (AU) relative to control (control =100), after normalization to expression of the TATA- binding protein (TBP...induced enterocolitis in zebrafish depends on the composition of the intestinal microbiota . Gastroenterology 137:1757-67 e1. 8. Fleming, A., Jankowski

  13. The importance of Zebrafish in biomedical research.

    PubMed

    Tavares, Bárbara; Santos Lopes, Susana

    2013-01-01

    Zebrafish (Danio rerio) is an ideal model organism for the study of vertebrate development. This is due to the large clutches that each couple produces, with up to 200 embryos every 7 days, and to the fact that the embryos and larvae are small, transparent and undergo rapid external development. Using scientific literature research tools available online and the keywords Zebrafish, biomedical research, human disease, and drug screening, we reviewed original studies and reviews indexed in PubMed. In this review we summarized work conducted with this model for the advancement of our knowledge related to several human diseases. We also focused on the biomedical research being performed in Portugal with the zebrafish model. Powerful live imaging and genetic tools are currently available for zebrafish making it a valuable model in biomedical research. The combination of these properties with the optimization of automated systems for drug screening has transformed the zebrafish into "a top model" in biomedical research, drug discovery and toxicity testing. Furthermore, with the optimization of xenografts technology it will be possible to use zebrafish to aide in the choice of the best therapy for each patient. Zebrafish is an excellent model organism in biomedical research, drug development and in clinical therapy.

  14. Zebrafish in hematology: sushi or science?

    PubMed Central

    Carradice, Duncan

    2008-01-01

    After a decade of the “modern era” of zebrafish hematology research, what have been their major contributions to hematology and what challenges does the model face? This review argues that, in hematology, zebrafish have demonstrated their suitability, are proving their utility, have supplied timely and novel discoveries, and are poised for further significant contributions. It presents an overview of the anatomy, physiology, and genetics of zebrafish hematopoiesis underpinning their use in hematology research. Whereas reverse genetic techniques enable functional studies of particular genes of interest, forward genetics remains zebrafish's particular strength. Mutants with diverse and interesting hematopoietic defects are emerging from multiple genetic screens. Some mutants model hereditary blood diseases, occasionally leading to disease genes first; others provide insights into developmental hematology. Models of malignant hematologic disorders provide tools for drug-target and pharmaceutics discovery. Numerous transgenic zebrafish with fluorescently marked blood cells enable live-cell imaging of inflammatory responses and host-pathogen interactions previously inaccessible to direct observation in vivo, revealing unexpected aspects of leukocyte behavior. Zebrafish disease models almost uniquely provide a basis for efficient whole animal chemical library screens for new therapeutics. Despite some limitations and challenges, their successes and discovery potential mean that zebrafish are here to stay in hematology research. PMID:18182572

  15. Zebrafish in hematology: sushi or science?

    PubMed

    Carradice, Duncan; Lieschke, Graham J

    2008-04-01

    After a decade of the "modern era" of zebrafish hematology research, what have been their major contributions to hematology and what challenges does the model face? This review argues that, in hematology, zebrafish have demonstrated their suitability, are proving their utility, have supplied timely and novel discoveries, and are poised for further significant contributions. It presents an overview of the anatomy, physiology, and genetics of zebrafish hematopoiesis underpinning their use in hematology research. Whereas reverse genetic techniques enable functional studies of particular genes of interest, forward genetics remains zebrafish's particular strength. Mutants with diverse and interesting hematopoietic defects are emerging from multiple genetic screens. Some mutants model hereditary blood diseases, occasionally leading to disease genes first; others provide insights into developmental hematology. Models of malignant hematologic disorders provide tools for drug-target and pharmaceutics discovery. Numerous transgenic zebrafish with fluorescently marked blood cells enable live-cell imaging of inflammatory responses and host-pathogen interactions previously inaccessible to direct observation in vivo, revealing unexpected aspects of leukocyte behavior. Zebrafish disease models almost uniquely provide a basis for efficient whole animal chemical library screens for new therapeutics. Despite some limitations and challenges, their successes and discovery potential mean that zebrafish are here to stay in hematology research.

  16. Streptococcus-Zebrafish Model of Bacterial Pathogenesis

    PubMed Central

    Neely, Melody N.; Pfeifer, John D.; Caparon, Michael

    2002-01-01

    Due to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish, Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 103 CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that of S. iniae in farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogen Streptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease. PMID:12065534

  17. Animal Models of Tuberculosis: Zebrafish

    PubMed Central

    van Leeuwen, Lisanne M.; van der Sar, Astrid M.; Bitter, Wilbert

    2015-01-01

    Over the past decade the zebrafish (Danio rerio) has become an attractive new vertebrate model organism for studying mycobacterial pathogenesis. The combination of medium-throughput screening and real-time in vivo visualization has allowed new ways to dissect host pathogenic interaction in a vertebrate host. Furthermore, genetic screens on the host and bacterial sides have elucidated new mechanisms involved in the initiation of granuloma formation and the importance of a balanced immune response for control of mycobacterial pathogens. This article will highlight the unique features of the zebrafish–Mycobacterium marinum infection model and its added value for tuberculosis research. PMID:25414379

  18. 2017 Midwest Zebrafish Meeting Report.

    PubMed

    Sandquist, Elizabeth; Petersen, Sarah C; Smith, Cody J

    2017-09-07

    The 2017 Midwest Zebrafish meeting was held from June 16 to 18 at the University of Cincinnati, sponsored by the Cincinnati Children's Hospital Divisions of Developmental Biology, Molecular Cardiovascular Biology, and Gastroenterology, Hepatology, and Nutrition. The meeting, organized by Saulius Sumanas, Joshua Waxman, and Chunyue Yin, hosted >130 attendees from 16 different states. Scientific sessions were focused on morphogenesis, neural development, novel technologies, and disease models, with Steve Ekker, Stephen Potter, and Lila Solnica-Krezel presenting keynote talks. In this article, we highlight the results and emerging themes from the meeting.

  19. A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia

    PubMed Central

    Pazhakh, Vahid; Clark, Sharon; Keightley, M. Cristina; Lieschke, Graham J.

    2017-01-01

    Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life. PMID:28281657

  20. Biliary atresia: From Australia to the zebrafish.

    PubMed

    Davenport, Mark

    2016-02-01

    This review is based upon an invited lecture for the 52nd Annual Meeting of the British Association of Paediatric Surgeons, July 2015. The aetiology of biliary atresia (BA) is at best obscure, but it is probable that a number of causes or pathophysiological mechanisms may be involved leading to the final common phenotype we recognise clinically. By way of illustration, similar conditions to human BA are described, including biliary agenesis, which is the normal state and peculiar final pattern of bile duct development in the jawless fish, the lamprey. Furthermore, there have been remarkable outbreaks in the Australian outback of BA in newborn lambs whose mothers were exposed to and grazed upon a particular plant species (Dysphania glomulifera) during gestation. More recent work using a zebrafish model has isolated a toxic isoflavonoid, now named Biliatresone, thought to be responsible for these outbreaks. Normal development of the bile ducts is reviewed and parallels drawn with two clinical variants thought to definitively have their origins in intrauterine life: Biliary Atresia Splenic Malformation syndrome (BASM) and Cystic Biliary Atresia (CBA). For both variants there is sufficient clinical evidence, including associated anomalies and antenatal detection, respectively, to warrant their aetiological attribution as developmental BA. CMV IgM +ve associated BA is a further variant that appears separate with distinct clinical, histological, and immunohistochemical features. In these it seems possible that this involves perinatal obliteration of a normally formed duct system. Although still circumstantial, this evidence appears convincing enough to perhaps warrant a different treatment strategy. This then still leaves the most common (more than 60% in Western series) variant, now termed Isolated BA, whereby origins can only be alluded to.

  1. How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs

    PubMed Central

    Pinho, Brígida R; Santos, Miguel M; Fonseca-Silva, Anabela; Valentão, Patrícia; Andrade, Paula B; Oliveira, Jorge M A

    2013-01-01

    Background and Purpose Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone. Experimental Approach Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish versus mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays. Key Results Complex I and II inhibitors induced developmental abnormalities, but their submaximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anaemia when transiently present following primitive erythropoiesis. Atovaquone was over 1000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxybenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction. Conclusion and Implications This study characterizes pharmacologically induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further

  2. How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs.

    PubMed

    Pinho, Brígida R; Santos, Miguel M; Fonseca-Silva, Anabela; Valentão, Patrícia; Andrade, Paula B; Oliveira, Jorge M A

    2013-07-01

    Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone. Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish versus mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays. Complex I and II inhibitors induced developmental abnormalities, but their submaximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anaemia when transiently present following primitive erythropoiesis. Atovaquone was over 1000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxybenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction. This study characterizes pharmacologically induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further, it evidences zebrafish's potential for in vivo efficacy or toxicity screening of

  3. Sprouting Buds of Zebrafish Research in Malaysia: First Malaysia Zebrafish Disease Model Workshop.

    PubMed

    Okuda, Kazuhide Shaun; Tan, Pei Jean; Patel, Vyomesh

    2016-04-01

    Zebrafish is gaining prominence as an important vertebrate model for investigating various human diseases. Zebrafish provides unique advantages such as optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it a perfect complement to the murine model equivalent in biomedical research. Due to these advantages, researchers in Malaysia are starting to take notice and incorporate the zebrafish model into their research activities. However, zebrafish research in Malaysia is still in its infancy stage and many researchers still remain unaware of the full potential of the zebrafish model or have limited access to related tools and techniques that are widely utilized in many zebrafish laboratories worldwide. To overcome this, we organized the First Malaysia Zebrafish Disease Model Workshop in Malaysia that took place on 11th and 12th of November 2015. In this workshop, we showcased how the zebrafish model is being utilized in the biomedical field in international settings as well as in Malaysia. For this, notable international speakers and those from local universities known to be carrying out impactful research using zebrafish were invited to share some of the cutting edge techniques that are used in their laboratories that may one day be incorporated in the Malaysian scientific community.

  4. Waterborne Risperidone Decreases Stress Response in Zebrafish.

    PubMed

    Idalencio, Renan; Kalichak, Fabiana; Rosa, João Gabriel Santos; de Oliveira, Tiago Acosta; Koakoski, Gessi; Gusso, Darlan; Abreu, Murilo Sander de; Giacomini, Ana Cristina Varrone; Barcellos, Heloísa Helena de Alcântara; Piato, Angelo L; Barcellos, Leonardo José Gil

    2015-01-01

    The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish.

  5. Waterborne Risperidone Decreases Stress Response in Zebrafish

    PubMed Central

    Kalichak, Fabiana; Rosa, João Gabriel Santos; de Oliveira, Tiago Acosta; Koakoski, Gessi; Gusso, Darlan; de Abreu, Murilo Sander; Giacomini, Ana Cristina Varrone; Barcellos, Heloísa Helena de Alcântara

    2015-01-01

    The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish. PMID:26473477

  6. Zebrafish tracking using convolutional neural networks

    PubMed Central

    XU, Zhiping; Cheng, Xi En

    2017-01-01

    Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable. PMID:28211462

  7. Zebrafish tracking using convolutional neural networks

    NASA Astrophysics Data System (ADS)

    Xu, Zhiping; Cheng, Xi En

    2017-02-01

    Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

  8. Learning and memory in zebrafish larvae.

    PubMed

    Roberts, Adam C; Bill, Brent R; Glanzman, David L

    2013-01-01

    Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebrafish that are likely to contribute major insights into the processes that underlie learning and memory.

  9. Automated measurement of zebrafish larval movement

    PubMed Central

    Cario, Clinton L; Farrell, Thomas C; Milanese, Chiara; Burton, Edward A

    2011-01-01

    Abstract The zebrafish is a powerful vertebrate model that is readily amenable to genetic, pharmacological and environmental manipulations to elucidate the molecular and cellular basis of movement and behaviour. We report software enabling automated analysis of zebrafish movement from video recordings captured with cameras ranging from a basic camcorder to more specialized equipment. The software, which is provided as open-source MATLAB functions, can be freely modified and distributed, and is compatible with multiwell plates under a wide range of experimental conditions. Automated measurement of zebrafish movement using this technique will be useful for multiple applications in neuroscience, pharmacology and neuropsychiatry. PMID:21646414

  10. Learning and memory in zebrafish larvae

    PubMed Central

    Roberts, Adam C.; Bill, Brent R.; Glanzman, David L.

    2013-01-01

    Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebrafish that are likely to contribute major insights into the processes that underlie learning and memory. PMID:23935566

  11. Toxicity of silver nanoparticles in zebrafish models.

    PubMed

    Asharani, P V; Lian Wu, Yi; Gong, Zhiyuan; Valiyaveettil, Suresh

    2008-06-25

    This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). Using starch and bovine serum albumin (BSA) as capping agents, silver nanoparticles were synthesized to study their deleterious effects and distribution pattern in zebrafish embryos (Danio rerio). Toxicological endpoints like mortality, hatching, pericardial edema and heart rate were recorded. A concentration-dependent increase in mortality and hatching delay was observed in Ag-np treated embryos. Additionally, nanoparticle treatments resulted in concentration-dependent toxicity, typified by phenotypes that had abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. Ag(+) ions and stabilizing agents showed no significant defects in developing embryos. Transmission electron microscopy (TEM) of the embryos demonstrated that nanoparticles were distributed in the brain, heart, yolk and blood of embryos as evident from the electron-dispersive x-ray analysis (EDS). Furthermore, the acridine orange staining showed an increased apoptosis in Ag-np treated embryos. These results suggest that silver nanoparticles induce a dose-dependent toxicity in embryos, which hinders normal development.

  12. Toxicity of silver nanoparticles in zebrafish models

    NASA Astrophysics Data System (ADS)

    Asharani, P. V.; Lian Wu, Yi; Gong, Zhiyuan; Valiyaveettil, Suresh

    2008-06-01

    This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). Using starch and bovine serum albumin (BSA) as capping agents, silver nanoparticles were synthesized to study their deleterious effects and distribution pattern in zebrafish embryos (Danio rerio). Toxicological endpoints like mortality, hatching, pericardial edema and heart rate were recorded. A concentration-dependent increase in mortality and hatching delay was observed in Ag-np treated embryos. Additionally, nanoparticle treatments resulted in concentration-dependent toxicity, typified by phenotypes that had abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. Ag+ ions and stabilizing agents showed no significant defects in developing embryos. Transmission electron microscopy (TEM) of the embryos demonstrated that nanoparticles were distributed in the brain, heart, yolk and blood of embryos as evident from the electron-dispersive x-ray analysis (EDS). Furthermore, the acridine orange staining showed an increased apoptosis in Ag-np treated embryos. These results suggest that silver nanoparticles induce a dose-dependent toxicity in embryos, which hinders normal development.

  13. Chromatin modification in zebrafish development.

    PubMed

    Cayuso Mas, Jordi; Noël, Emily S; Ober, Elke A

    2011-01-01

    The generation of complex organisms requires that an initial population of cells with identical gene expression profiles can adopt different cell fates during development by progressively diverging transcriptional programs. These programs depend on the binding of transcritional regulators to specific genomic sites, which in turn is controlled by modifications of the chromatin. Chromatin modifications may occur directly upon DNA by methylation of specific nucleotides, or may involve post-translational modification of histones. Local regulation of histone post-translational modifications regionalizes the genome into euchromatic regions, which are more accessible to DNA-binding factors, and condensed heterochromatic regions, inhibiting the binding of such factors. In addition, these modifications may be required in a genome-wide fashion for processes such as DNA replication or chromosome condensation. From an embryologist's point of view chromatin modifications are intensively studied in the context of imprinting and have more recently received increasing attention in understanding the basis of pluripotency and cellular differentiation. Here, we describe recently uncovered roles of chromatin modifications in zebrafish development and regeneration, as well as available resources and commonly used techniques. We provide a general introduction into chromatin modifications and their respective functions with a focus on gene transcription, as well as key aspects of their roles in the early zebrafish embryo, neural development, formation of the digestive system and tissue regeneration.

  14. Optimizing synchrotron microCT for high-throughput phenotyping of zebrafish

    NASA Astrophysics Data System (ADS)

    La Rivière, Patrick J.; Clark, Darin; Rojek, Alexandra; Vargas, Phillip; Xiao, Xianghui; DeCarlo, Francesco; Kindlmann, Gordon; Cheng, Keith

    2010-09-01

    We are creating a state-of-the-art 2D and 3D imaging atlas of zebrafish development. The atlas employs both 2D histology slides and 3D benchtop and synchrotron micro CT results. Through this atlas, we expect to document normal and abnormal organogenesis, to reveal new levels of structural detail, and to advance image informatics as a form of systems biology. The zebrafish has become a widely used model organism in biological and biomedical research for studies of vertebrate development and gene function. In this work, we will report on efforts to optimize synchrotron microCT imaging parameters for zebrafish at crucial developmental stages. The aim of these studies is to establish protocols for high-throughput phenotyping of normal, mutant and diseased zebrafish. We have developed staining and embedding protocols using different heavy metal stains (osmium tetroxide and uranyl acetate) and different embedding media (Embed 812 and glycol methacrylate). We have explored the use of edge subtraction and multi-energy techniques for contrast enhancement and we have examined the use of different sample-detector distances with unstained samples to explore and optimize phase-contrast enhancement effects. We will report principally on our efforts to optimize energy choice for single- and multi-energy studies as well as our efforts to optimize the degree of phase contrast enhancement.

  15. Investigations of photoreceptor synaptic transmission and light adaptation in the zebrafish visual mutant nrc.

    PubMed

    Van Epps, H A; Yim, C M; Hurley, J B; Brockerhoff, S E

    2001-03-01

    To characterize the retinal physiology of the zebrafish visual mutant no optokinetic response c (nrc) and to identify the genetic map position of the nrc mutation. Electroretinograms were recorded from wild-type and nrc zebrafish larvae between 5 to 6 days postfertilization. Responses to flash stimuli, On and Off responses to prolonged light stimuli, and responses to flash stimuli with constant background illumination were characterized. The glutamate agonist, 2-amino-4-phosphonobutyric acid (APB) was used to examine the photoreceptor specific a-wave component of the electroretinogram. Amplified fragment length polymorphism methodology was used to place the nrc mutation on the zebrafish genomic map. nrc and wild-type zebrafish larvae 5 to 6 days postfertilization have similar threshold responses to light, but the b-wave of the nrc electroretinogram is significantly delayed and reduced in amplitude. On and Off responses of nrc larvae to prolonged light have multiple oscillations that do not occur in normal zebrafish larvae after 5 days postfertilization. Analysis of the b-wave demonstrated a light adaptation defect in nrc that causes saturation at background light levels approximately 1 order of magnitude less than those with wild-type larvae. Application of the glutamate analog, APB, uncovered the photoreceptor component of the electroretinogram and revealed a light adaptation defect in nrc photoreceptors. The nrc mutation was placed approximately 0.2 cM from sequence length polymorphism marker Z7504 on linkage group 10. The zebrafish mutant nrc is a possible model for human retinal disease. nrc has defects in photoreceptor synaptic transmission and light adaptation. The nrc mutant phenotype shows striking similarities with phenotypes of dystrophin glycoprotein complex mutants, including patients with Duchenne/Becker muscular dystrophy. Localization of the nrc mutation now makes it possible to evaluate candidate genes and clone the nrc gene.

  16. A Study of the Adult Zebrafish Ventricular Function by Retrospective Doppler-Gated Ultrahigh-Frame-Rate Echocardiography

    PubMed Central

    Liu, Ting-Yu; Lee, Po-Yang; Huang, Chih-Chung; Sun, Lei; Shung, K. Kirk

    2014-01-01

    The zebrafish (Danio rerio) has become a preferred animal model for studying various human diseases, particularly those related to cardiovascular regeneration; therefore, a noninvasive imaging modality is needed for observing the cardiac function of zebrafish. Because of its high resolution, high-frequency ultrasound B-mode imaging has recently been used successfully to observe the heart of adult zebrafish. However, ultrahigh-frame-rate echocardiography combining B-mode imaging and color flow imaging is still needed to observe the detailed transient motions of the zebrafish ventricle. This study develops an 80-MHz ultrahigh-frame-rate echocardiography system for this purpose, based on retrospective Doppler- gated technology. B-mode and color flow images of the cardiovascular system of the zebrafish were reconstructed by two-dimensional autocorrelation at maximum frame rates of up to 40 000 and 400 fps, respectively. The timings of end diastole (ED) and end systole (ES) of ventricle can be determined by using this high-resolution image system. Two ventricular function parameters—fractional shortening (FS) and fractional area change (FAC)—were measured for evaluating the ventricular function by using ED and ES with their corresponding ventricular dimensions. The experimental results indicated that the measured FS values were 42 ± 4% (mean ± standard deviation) and 60 ± 13% for the long axis and short axis of the ventricle, respectively, and that FAC was 77 ± 9%. This is the first report of these ventricular function parameters for a normal adult zebrafish. The results showed that retrospective high-frequency echocardiography is a useful tool for studying the cardiac function of normal adult zebrafish. PMID:24658716

  17. Modeling Syndromic Congenital Heart Defects in Zebrafish.

    PubMed

    Grant, Meagan G; Patterson, Victoria L; Grimes, Daniel T; Burdine, Rebecca D

    2017-01-01

    Cardiac development is a dynamic process regulated by spatial and temporal cues that are integrated to effect molecular, cellular, and tissue-level events that form the adult heart. Disruption of these highly orchestrated events can be devastating for cardiac form and function. Aberrations in heart development result in congenital heart defects (CHDs), which affect 1 in 100 infants in the United States each year. Zebrafish have proven informative as a model organism to understand both heart development and the mechanisms associated with CHDs due to the similarities in heart morphogenesis among vertebrates, as well as their genetic tractability and amenability to live imaging. In this review, we discuss the mechanisms of zebrafish heart development and the utility of zebrafish for understanding syndromic CHDs, those cardiac abnormalities that occur in the context of multisystem disorders. We conclude with avenues of zebrafish research that will potentially inform future therapeutic approaches for the treatment of CHDs.

  18. Behavioral screening for neuroactive drugs in zebrafish.

    PubMed

    Rihel, Jason; Schier, Alexander F

    2012-03-01

    The larval zebrafish has emerged asa vertebrate model system amenable to small molecule screens for probing diverse biological pathways. Two large-scale small molecule screens examined the effects of thousands of drugs on larval zebrafish sleep/wake and photomotor response behaviors. Both screens identified hundreds of molecules that altered zebrafish behavior in distinct ways. The behavioral profiles induced by these small molecules enabled the clustering of compounds according to shared phenotypes. This approach identified regulators of sleep/wake behavior and revealed the biological targets for poorly characterized compounds. Behavioral screening for neuroactive small molecules in zebrafish is an attractive complement to in vitro screening efforts, because the complex interactions in the vertebrate brain can only be revealed in vivo.

  19. Zebrafish Models for Human Acute Organophosphorus Poisoning

    PubMed Central

    Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J.; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick II, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B. Lynn; Zorzano, Antonio; Soares, Amadeu M.V.M; Raldúa, Demetrio

    2015-01-01

    Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning. PMID:26489395

  20. Zebrafish Models for Human Acute Organophosphorus Poisoning.

    PubMed

    Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick Ii, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B Lynn; Zorzano, Antonio; Soares, Amadeu M V M; Raldúa, Demetrio

    2015-10-22

    Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

  1. Episodic-like memory in zebrafish.

    PubMed

    Hamilton, Trevor J; Myggland, Allison; Duperreault, Erika; May, Zacnicte; Gallup, Joshua; Powell, Russell A; Schalomon, Melike; Digweed, Shannon M

    2016-11-01

    Episodic-like memory tests often aid in determining an animal's ability to recall the what, where, and which (context) of an event. To date, this type of memory has been demonstrated in humans, wild chacma baboons, corvids (Scrub jays), humming birds, mice, rats, Yucatan minipigs, and cuttlefish. The potential for this type of memory in zebrafish remains unexplored even though they are quickly becoming an essential model organism for the study of a variety of human cognitive and mental disorders. Here we explore the episodic-like capabilities of zebrafish (Danio rerio) in a previously established mammalian memory paradigm. We demonstrate that when zebrafish were presented with a familiar object in a familiar context but a novel location within that context, they spend more time in the novel quadrant. Thus, zebrafish display episodic-like memory as they remember what object they saw, where they saw it (quadrant location), and on which occasion (yellow or blue walls) it was presented.

  2. Histocompatibility and Hematopoietic Transplantation in the Zebrafish

    PubMed Central

    de Jong, Jill L. O.; Zon, Leonard I.

    2012-01-01

    The zebrafish has proven to be an excellent model for human disease, particularly hematopoietic diseases, since these fish make similar types of blood cells as humans and other mammals. The genetic program that regulates the development and differentiation of hematopoietic cells is highly conserved. Hematopoietic stem cells (HSCs) are the source of all the blood cells needed by an organism during its lifetime. Identifying an HSC requires a functional assay, namely, a transplantation assay consisting of multilineage engraftment of a recipient and subsequent serial transplant recipients. In the past decade, several types of hematopoietic transplant assays have been developed in the zebrafish. An understanding of the major histocompatibility complex (MHC) genes in the zebrafish has lagged behind transplantation experiments, limiting the ability to perform unbiased competitive transplantation assays. This paper summarizes the different hematopoietic transplantation experiments performed in the zebrafish, both with and without immunologic matching, and discusses future directions for this powerful experimental model of human blood diseases. PMID:22778744

  3. Zebrafish as a model for systems biology.

    PubMed

    Mushtaq, Mian Yahya; Verpoorte, Robert; Kim, Hye Kyong

    2013-01-01

    Zebrafish offer a unique vertebrate model for research areas such as drug development, disease modeling and other biological exploration. There is significant conservation of genetics and other cellular networks among zebrafish and other vertebrate models, including humans. Here we discuss the recent work and efforts made in different fields of biology to explore the potential of zebrafish. Along with this, we also reviewed the concept of systems biology. A biological system is made up of a large number of components that interact in a huge variety of combinations. To understand completely the behavior of a system, it is important to know its components and interactions, and this can be achieved through a systems biology approach. At the end of the paper we present a concept of integrating zebrafish into the systems biology approach.

  4. Polygenic Sex Determination System in Zebrafish

    PubMed Central

    Liew, Woei Chang; Bartfai, Richard; Lim, Zijie; Sreenivasan, Rajini; Siegfried, Kellee R.; Orban, Laszlo

    2012-01-01

    Background Despite the popularity of zebrafish as a research model, its sex determination (SD) mechanism is still unknown. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available. Recent publications suggest that environmental factors within the natural range have minimal impact on sex ratios of zebrafish populations. The primary aim of this study is to find out more about how sex is determined in zebrafish. Methodology/Principal Findings Using classical breeding experiments, we found that sex ratios across families were wide ranging (4.8% to 97.3% males). On the other hand, repeated single pair crossings produced broods of very similar sex ratios, indicating that parental genotypes have a role in the sex ratio of the offspring. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. Further examinations by a PCR-based “blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. Together with the ability to increase the sex bias of lines by selective breeding, these data suggest that zebrafish is unlikely to utilize a chromosomal sex determination (CSD) system. Conclusions/Significance Taken together, our study suggests that zebrafish sex is genetically determined with limited, secondary influences from the environment. As we have not found any sign for CSD in the species, we propose that the zebrafish has a polygenic sex determination system. PMID:22506019

  5. Spinal cord transection in the larval zebrafish.

    PubMed

    Briona, Lisa K; Dorsky, Richard I

    2014-05-21

    Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

  6. Age- and size-related changes in the inner ear and hearing ability of the adult zebrafish (Danio rerio).

    PubMed

    Higgs, Dennis M; Souza, Marcy J; Wilkins, Heather R; Presson, Joelle C; Popper, Arthur N

    2002-06-01

    Fishes, unlike most other vertebrate groups, continue to add sensory hair cells to their ears for much of their lives. However, it is not clear whether the addition ever stops or how the addition of sensory cells impacts hearing ability. In this article, we tested both questions using the zebrafish, Danio rerio. Our results not only have important implications for understanding the consequences of adding sensory receptors, but these results for normal zebrafish also serve as valuable baseline information for future studies of select mutations on the ear and hearing of this species. Our results show that hair cell production continues in uncrowded zebrafish up to 10 months of age (about one-third of a normal life span), but despite this addition there is no change in hearing sensitivity or bandwidth. Therefore, hearing is not related to the number of sensory cells in the ear in juvenile and adult animals. We also show that despite no net addition of hair cells after about 10 months, hair cells are still being produced, but at a lower rate, presumably to replace cells that are dying. Moreover, crowding of zebrafish has a marked impact on the growth of the fish and on the addition of sensory cells to the ear. We also demonstrate that fish size, not age, is a better indicator of developmental state of zebrafish.

  7. Retinal Regeneration Following OCT-Guided Laser Injury in Zebrafish

    PubMed Central

    DiCicco, Rose M.; Bell, Brent A.; Kaul, Charles; Hollyfield, Joe G.; Anand-Apte, Bela; Perkins, Brian D.; Tao, Yuankai K.; Yuan, Alex

    2014-01-01

    Purpose. Establish a focal injury/regeneration model in zebrafish using laser photocoagulation guided by optical coherence tomography (OCT). Methods. Adult zebrafish were imaged by OCT and confocal scanning laser ophthalmoscopy (cSLO) in room air through a contact lens. Using a beam combiner, 532-nm laser photocoagulation was applied using the OCT C-scan image for targeting. Laser spots of 42 to 47 mW were delivered to the retina. At multiple intervals post injury, fish were imaged using both OCT and cSLO to follow the progression of each lesion. Histologic sections and TUNEL staining were performed to monitor the injury response. Results. Round lesions (26057 ± 621 μm2) localized to the outer retina were successfully applied. Laser application was visualized by real-time OCT and lesions were detectable by both OCT and cSLO in vivo. Lesion size increased 1 day post lesion then decreased in size. Histologic sections showed focal areas of damage localized primarily to the outer retina. By 3 weeks, the damaged areas had regenerated and a fully laminated structure was re-established. However, subtle changes can still be detected by OCT, cSLO imaging, and histology. Infrared darkfield imaging was more sensitive than OCT at revealing subtle changes in regenerated areas. Conclusions. Optical coherence tomography–guided laser photocoagulation is a useful tool for inducing localized lesions and studying retinal regeneration in zebrafish. This novel method will allow us to characterize the cellular and molecular changes that take place at the interface between normal and damaged tissue. Regeneration can be observed using high-resolution OCT and cSLO imaging in vivo. PMID:25205862

  8. Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration

    PubMed Central

    Chen, William C. W.; Wang, Zhouguang; Missinato, Maria Azzurra; Park, Dae Woo; Long, Daniel Ward; Liu, Heng-Jui; Zeng, Xuemei; Yates, Nathan A.; Kim, Kang; Wang, Yadong

    2016-01-01

    Heart attack is a global health problem that leads to significant morbidity, mortality, and health care burden. Adult human hearts have very limited regenerative capability after injury. However, evolutionarily primitive species generally have higher regenerative capacity than mammals. The extracellular matrix (ECM) may contribute to this difference. Mammalian cardiac ECM may not be optimally inductive for cardiac regeneration because of the fibrotic, instead of regenerative, responses in injured adult mammalian hearts. Given the high regenerative capacity of adult zebrafish hearts, we hypothesize that decellularized zebrafish cardiac ECM (zECM) made from normal or healing hearts can induce mammalian heart regeneration. Using zebrafish and mice as representative species of lower vertebrates and mammals, we show that a single administration of zECM, particularly the healing variety, enables cardiac functional recovery and regeneration of adult mouse heart tissues after acute myocardial infarction. zECM-treated groups exhibit proliferation of the remaining cardiomyocytes and multiple cardiac precursor cell populations and reactivation of ErbB2 expression in cardiomyocytes. Furthermore, zECM exhibits pro-proliferative and chemotactic effects on human cardiac precursor cell populations in vitro. These contribute to the structural preservation and correlate with significantly higher cardiac contractile function, notably less left ventricular dilatation, and substantially more elastic myocardium in zECM-treated hearts than control animals treated with saline or decellularized adult mouse cardiac ECM. Inhibition of ErbB2 activity abrogates beneficial effects of zECM administration, indicating the possible involvement of ErbB2 signaling in zECM-mediated regeneration. This study departs from conventional focuses on mammalian ECM and introduces a new approach for cardiac tissue regeneration. PMID:28138518

  9. Zebrafish heart as a model for human cardiac electrophysiology.

    PubMed

    Vornanen, Matti; Hassinen, Minna

    2016-01-01

    The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.

  10. Can Zebrafish be used to Identify Developmentally Neurotoxic Chemicals

    EPA Science Inventory

    Can Zebrafish be Used to Identify Developmentally Neurotoxic Chemicals? The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental neurotoxicity. We are exploring behavioral methods using zebrafish by desig...

  11. Can Zebrafish be used to Identify Developmentally Neurotoxic Chemicals

    EPA Science Inventory

    Can Zebrafish be Used to Identify Developmentally Neurotoxic Chemicals? The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental neurotoxicity. We are exploring behavioral methods using zebrafish by desig...

  12. Zebrafish models of cerebrovascular disease.

    PubMed

    Walcott, Brian P; Peterson, Randall T

    2014-04-01

    Perturbations in cerebral blood flow and abnormalities in blood vessel structure are the hallmarks of cerebrovascular disease. While there are many genetic and environmental factors that affect these entities through a heterogeneous group of disease processes, the ultimate final pathologic insult in humans is defined as a stroke, or damage to brain parenchyma. In the case of ischemic stroke, blood fails to reach its target destination whereas in hemorrhagic stroke, extravasation of blood occurs outside of the blood vessel lumen, resulting in direct damage to brain parenchyma. As these acute events can be neurologically devastating, if not fatal, development of novel therapeutics are urgently needed. The zebrafish (Danio rerio) is an attractive model for the study of cerebrovascular disease because of its morphological and physiological similarity to human cerebral vasculature, its ability to be genetically manipulated, and its fecundity allowing for large-scale, phenotype-based screens.

  13. Neuroblastoma and Its Zebrafish Model.

    PubMed

    Zhu, Shizhen; Thomas Look, A

    2016-01-01

    Neuroblastoma, an important developmental tumor arising in the peripheral sympathetic nervous system (PSNS), accounts for approximately 10 % of all cancer-related deaths in children. Recent genomic analyses have identified a spectrum of genetic alterations in this tumor. Amplification of the MYCN oncogene is found in 20 % of cases and is often accompanied by mutational activation of the ALK (anaplastic lymphoma kinase) gene, suggesting their cooperation in tumor initiation and spread. Understanding how complex genetic changes function together in oncogenesis has been a continuing and daunting task in cancer research. This challenge was addressed in neuroblastoma by generating a transgenic zebrafish model that overexpresses human MYCN and activated ALK in the PSNS, leading to tumors that closely resemble human neuroblastoma and new opportunities to probe the mechanisms that underlie the pathogenesis of this tumor. For example, coexpression of activated ALK with MYCN in this model triples the penetrance of neuroblastoma and markedly accelerates tumor onset, demonstrating the interaction of these modified genes in tumor development. Further, MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. In the context of MYCN overexpression, activated ALK provides prosurvival signals that block this apoptotic response, allowing continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma. This application of the zebrafish model illustrates its value in rational assessment of the multigenic changes that define neuroblastoma pathogenesis and points the way to future studies to identify novel targets for therapeutic intervention.

  14. Loss of the small heat shock protein αA-crystallin does not lead to detectable defects in early zebrafish lens development.

    PubMed

    Posner, Mason; Skiba, Jackie; Brown, Mary; Liang, Jennifer O; Nussbaum, Justin; Prior, Heather

    2013-11-01

    Alpha crystallins are small heat shock proteins essential to normal ocular lens function. They also help maintain homeostasis in many non-ocular vertebrate tissues and their expression levels change in multiple diseases of the nervous and cardiovascular system and during cancer. The specific roles that α-crystallins may play in eye development are unclear. Studies with knockout mice suggested that only one of the two mammalian α-crystallins is required for normal early lens development. However, studies in two fish species suggested that reduction of αA-crystallin alone could inhibit normal fiber cell differentiation, cause cataract and contribute to lens degeneration. In this study we used synthetic antisense morpholino oligomers to suppress the expression of zebrafish αA-crystallin to directly test the hypothesis that, unlike mammals, the zebrafish requires αA-crystallin for normal early lens development. Despite the reduction of zebrafish αA-crystallin protein to undetectable levels by western analysis through 4 days of development we found no changes in fiber cell differentiation, lens morphology or transparency. In contrast, suppression of AQP0a expression, previously shown to cause lens cataract, produced irregularly shaped lenses, delay in fiber cell differentiation and lens opacities detectable by confocal microscopy. The normal development observed in αA-crystallin deficient zebrafish embryos may reflect similarly non-essential roles for this protein in the early stages of both zebrafish and mammalian lens development. This finding has ramifications for a growing number of researchers taking advantage of the zebrafish's transparent external embryos to study vertebrate eye development. Our demonstration that lens cataracts can be visualized in three-dimensions by confocal microscopy in a living zebrafish provides a new tool for studying the causes, development and prevention of lens opacities.

  15. Chemical screening in zebrafish for novel biological and therapeutic discovery

    PubMed Central

    Wiley, D.S.; Redfield, S.E.; Zon, L.I.

    2017-01-01

    Zebrafish chemical screening allows for an in vivo assessment of small molecule modulation of biological processes. Compound toxicities, chemical alterations by metabolism, pharmacokinetic and pharmacodynamic properties, and modulation of cell niches can be studied with this method. Furthermore, zebrafish screening is straightforward and cost effective. Zebrafish provide an invaluable platform for novel therapeutic discovery through chemical screening. PMID:28129862

  16. Viral diseases in zebrafish: what is known and unknown.

    PubMed

    Crim, Marcus J; Riley, Lela K

    2012-01-01

    Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model.

  17. Viral Diseases in Zebrafish: What Is Known and Unknown

    PubMed Central

    Crim, Marcus J.; Riley, Lela K.

    2013-01-01

    Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model. PMID:23382345

  18. In Vivo Quantitative Study of Sized-Dependent Transport and Toxicity of Single Silver Nanoparticles Using Zebrafish Embryos

    PubMed Central

    Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Desai, Tanvi; Cherukui, Pavan K.; Xu, Xiao-Hong Nancy

    2012-01-01

    Nanomaterials possess distinctive physicochemical properties (e.g., small sizes, high surface area-to-volume ratios) and promise a wide variety of applications, ranging from design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advance in nanotechnology. In this study, we have synthesized and characterized purified and stable (non-aggregation) silver nanoparticles (Ag NPs, 41.6±9.1 nm in average diameters), and utilized early-developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe diffusion and toxicity of Ag NPs. We found that single Ag NPs (30–72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose and size dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤ 0.02 nM), 75–91% of embryos developed to normal zebrafish. At the higher concentrations of NPs (≥ 0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02–0.2 nM), embryos developed to various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish, and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6±3.5 nm), the results further demonstrate striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6±9.1 nm) are more toxic than the smaller Ag NPs (11.6±3.5 nm). PMID:22486336

  19. Microanatomy of adult zebrafish extraocular muscles.

    PubMed

    Kasprick, Daniel S; Kish, Phillip E; Junttila, Tyler L; Ward, Lindsay A; Bohnsack, Brenda L; Kahana, Alon

    2011-01-01

    Binocular vision requires intricate control of eye movement to align overlapping visual fields for fusion in the visual cortex, and each eye is controlled by 6 extraocular muscles (EOMs). Disorders of EOMs are an important cause of symptomatic vision loss. Importantly, EOMs represent specialized skeletal muscles with distinct gene expression profile and susceptibility to neuromuscular disorders. We aim to investigate and describe the anatomy of adult zebrafish extraocular muscles (EOMs) to enable comparison with human EOM anatomy and facilitate the use of zebrafish as a model for EOM research. Using differential interference contrast (DIC), epifluorescence microscopy, and precise sectioning techniques, we evaluate the anatomy of zebrafish EOM origin, muscle course, and insertion on the eye. Immunofluorescence is used to identify components of tendons, basement membrane and neuromuscular junctions (NMJs), and to analyze myofiber characteristics. We find that adult zebrafish EOM insertions on the globe parallel the organization of human EOMs, including the close proximity of specific EOM insertions to one another. However, analysis of EOM origins reveals important differences between human and zebrafish, such as the common rostral origin of both oblique muscles and the caudal origin of the lateral rectus muscles. Thrombospondin 4 marks the EOM tendons in regions that are highly innervated, and laminin marks the basement membrane, enabling evaluation of myofiber size and distribution. The NMJs appear to include both en plaque and en grappe synapses, while NMJ density is much higher in EOMs than in somatic muscles. In conclusion, zebrafish and human EOM anatomy are generally homologous, supporting the use of zebrafish for studying EOM biology. However, anatomic differences exist, revealing divergent evolutionary pressures.

  20. Single epicardial cell transcriptome sequencing identifies Caveolin 1 as an essential factor in zebrafish heart regeneration

    PubMed Central

    Cao, Jingli; Navis, Adam; Cox, Ben D.; Dickson, Amy L.; Gemberling, Matthew; Karra, Ravi; Bagnat, Michel; Poss, Kenneth D.

    2016-01-01

    In contrast to mammals, adult zebrafish have a high capacity to regenerate damaged or lost myocardium through proliferation of cardiomyocytes spared from damage. The epicardial sheet covering the heart is activated by injury and aids muscle regeneration through paracrine effects and as a multipotent cell source, and has received recent attention as a target in cardiac repair strategies. Although it is recognized that epicardium is required for muscle regeneration and itself has high regenerative potential, the extent of cellular heterogeneity within epicardial tissue is largely unexplored. Here, we performed transcriptome analysis on dozens of epicardial lineage cells purified from zebrafish harboring a transgenic reporter for the pan-epicardial gene tcf21. Hierarchical clustering analysis suggested the presence of at least three epicardial cell subsets defined by expression signatures. We validated many new pan-epicardial and epicardial markers by alternative expression assays. Additionally, we explored the function of the scaffolding protein and main component of caveolae, caveolin 1 (cav1), which was present in each epicardial subset. In BAC transgenic zebrafish, cav1 regulatory sequences drove strong expression in ostensibly all epicardial cells and in coronary vascular endothelial cells. Moreover, cav1 mutant zebrafish generated by genome editing showed grossly normal heart development and adult cardiac anatomy, but displayed profound defects in injury-induced cardiomyocyte proliferation and heart regeneration. Our study defines a new platform for the discovery of epicardial lineage markers, genetic tools, and mechanisms of heart regeneration. PMID:26657776

  1. Transmission electron microscopic evaluation of neuronal changes in methylmercury-exposed zebrafish embryos (Danio rerio).

    PubMed

    Hassan, Said A; Farouk, Sameh M; Abbott, Louise C

    2016-01-01

    Our work aimed to elucidate the ultrastructural changes associated with brain neurons in wild-type zebrafish embryos exposed to different concentrations of methylmercury. Zebrafish embryos were exposed to one of five concentrations of methylmercury (0 [negative control], 5, 10, 50, and 80 parts per billion) starting at six hours post fertilization (hpf). At 96 hpf, cells in the zebrafish embryo brains were examined using transmission electron microscopy. The developing neurons of the control embryos sowed normal cellular ultrastructure. Few alterations were observed among the neurons of zebrafish embryos exposed to 5 ppb methylmercury. The cells of the embryos exposed to 10 ppb methylmercury showed slight cellular degeneration as demonstrated by the accumulation of electron dens bodies which were presumably lysosomes in different stages of formation. In embryos exposed to 50 ppb methylmercury, the neuronal cytoplasm conained large electron dense lysosomes and the rough endoplasmic reticulum appeared to be reduced and irregular in shape. Furthermore, the embryonic brain neurons exposed to 80 ppb methylmercury showed the most severe ultrastructural changes, including some that were consistent with different stages of the cell death process. Obvious cellular changes were observed in this highest exposure group included: disrupted or degenerating nuclei; fragmentation or vacuolization of mitochondrial cristae; and loss of mitochondrial matrix density. Based on these observations, we conclude that these different morphological patterns of cellular changes may reflect either different stages of the cell death process or different types of cell death due to 24 hours of exposure to 80 ppb methylmercury.

  2. Perturbation of cytosolic calcium by 2-aminoethoxydiphenyl borate and caffeine affects zebrafish myofibril alignment.

    PubMed

    Wu, Hsin-Ju; Fong, Tsorng-Harn; Chen, Shen-Liang; Wei, Jen-Cheng; Wang, I-Jong; Wen, Chi-Chung; Chang, Chao-Yuan; Chen, Xing-Guang; Chen, Wei-Yu; Chen, Hui-Min; Horng, Juin-Lin; Wang, Yun-Hsin; Chen, Yau-Hung

    2015-03-01

    The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment. Copyright © 2014 John Wiley & Sons, Ltd.

  3. High-resolution tissue Doppler imaging of the zebrafish heart during its regeneration.

    PubMed

    Huang, Chih-Chung; Su, Ta-Han; Shih, Cho-Chiang

    2015-02-01

    The human heart cannot regenerate after injury, whereas the adult zebrafish can fully regenerate its heart even after 20% of the ventricle is amputated. Many studies have begun to reveal the cellular and molecular mechanisms underlying this regenerative process, which have exciting implications for human cardiac diseases. However, the dynamic functions of the zebrafish heart during regeneration are not yet understood. This study established a high-resolution echocardiography for tissue Doppler imaging (TDI) of the zebrafish heart to explore the cardiac functions during different regeneration phases. Experiments were performed on AB-line adult zebrafish (n=40) in which 15% of the ventricle was surgically removed. An 80-MHz ultrasound TDI based on color M-mode imaging technology was employed. The cardiac flow velocities and patterns from both the ventricular chamber and myocardium were measured at different regeneration phases relative to the day of amputation. The peak velocities of early diastolic inflow, early diastolic myocardial motion, late diastolic myocardial motion, early diastolic deceleration slope, and heart rate were increased at 3 days after the myocardium amputation, but these parameters gradually returned to close to their baseline values for the normal heart at 7 days after amputation. The peak velocities of late diastolic inflow, ventricular systolic outflow, and systolic myocardial motion did not significantly differ during the heart regeneration.

  4. High-Resolution Tissue Doppler Imaging of the Zebrafish Heart During Its Regeneration

    PubMed Central

    Su, Ta-Han; Shih, Cho-Chiang

    2015-01-01

    Abstract The human heart cannot regenerate after injury, whereas the adult zebrafish can fully regenerate its heart even after 20% of the ventricle is amputated. Many studies have begun to reveal the cellular and molecular mechanisms underlying this regenerative process, which have exciting implications for human cardiac diseases. However, the dynamic functions of the zebrafish heart during regeneration are not yet understood. This study established a high-resolution echocardiography for tissue Doppler imaging (TDI) of the zebrafish heart to explore the cardiac functions during different regeneration phases. Experiments were performed on AB-line adult zebrafish (n=40) in which 15% of the ventricle was surgically removed. An 80-MHz ultrasound TDI based on color M-mode imaging technology was employed. The cardiac flow velocities and patterns from both the ventricular chamber and myocardium were measured at different regeneration phases relative to the day of amputation. The peak velocities of early diastolic inflow, early diastolic myocardial motion, late diastolic myocardial motion, early diastolic deceleration slope, and heart rate were increased at 3 days after the myocardium amputation, but these parameters gradually returned to close to their baseline values for the normal heart at 7 days after amputation. The peak velocities of late diastolic inflow, ventricular systolic outflow, and systolic myocardial motion did not significantly differ during the heart regeneration. PMID:25517185

  5. Stat3/Cdc25a-dependent cell proliferation promotes embryonic axis extension during zebrafish gastrulation

    PubMed Central

    Sepich, Diane S.

    2017-01-01

    Cell proliferation has generally been considered dispensable for anteroposterior extension of embryonic axis during vertebrate gastrulation. Signal transducer and activator of transcription 3 (Stat3), a conserved controller of cell proliferation, survival and regeneration, is associated with human scoliosis, cancer and Hyper IgE Syndrome. Zebrafish Stat3 was proposed to govern convergence and extension gastrulation movements in part by promoting Wnt/Planar Cell Polarity (PCP) signaling, a conserved regulator of mediolaterally polarized cell behaviors. Here, using zebrafish stat3 null mutants and pharmacological tools, we demonstrate that cell proliferation contributes to anteroposterior embryonic axis extension. Zebrafish embryos lacking maternal and zygotic Stat3 expression exhibit normal convergence movements and planar cell polarity signaling, but transient axis elongation defect due to insufficient number of cells resulting largely from reduced cell proliferation and increased apoptosis. Pharmacologic inhibition of cell proliferation during gastrulation phenocopied axis elongation defects. Stat3 regulates cell proliferation and axis extension in part via upregulation of Cdc25a expression during oogenesis. Accordingly, restoring Cdc25a expression in stat3 mutants partially suppressed cell proliferation and gastrulation defects. During later development, stat3 mutant zebrafish exhibit stunted growth, scoliosis, excessive inflammation, and fail to thrive, affording a genetic tool to study Stat3 function in vertebrate development, regeneration, and disease. PMID:28222105

  6. Influences of textured substrates on the heart rate of developing zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Yun; Chen, Chia-Yuan

    2013-07-01

    Identification of the effects of different textured substrates on zebrafish (Danio rerio) embryos provides insights into the influence of external stimuli on normal cardiovascular functions in the developmental stages of the embryos. This knowledge can be used in numerous genetic studies using zebrafish as an animal model as well as in bioanalytical assays using digital microfluidics. In this study, zebrafish embryos were systematically positioned and in vivo imaged on four types of silicon substrates. These substrates exhibited surface textures and surface wettability that were well modulated by wet chemical etching. The heart rate of the developing embryos significantly increased by 9.1% upon exposure to textured Si substrates with nanostructured surfaces compared with bare Si substrates. Modulation of surface wettability in the tested substrates also responded to the increase in the heart rate of the embryo; however, the effect of surface wettability on heart rate was slight compared with the effect of texture. In-depth experimental and statistical investigations of heart rate under the effects of substrate textures imply a pathway through which the inner mass of the embryo reacts to external stimuli. These findings contribute to zebrafish-related studies and suggest other factors to consider in the design of nanostructure-based microfluidics and other biomedical devices.

  7. Development of high-content assays for kidney progenitor cell expansion in transgenic zebrafish.

    PubMed

    Sanker, Subramaniam; Cirio, Maria Cecilia; Vollmer, Laura L; Goldberg, Natasha D; McDermott, Lee A; Hukriede, Neil A; Vogt, Andreas

    2013-12-01

    Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

  8. Effects of ZnSO4-induced peripheral anosmia on zebrafish behavior and physiology.

    PubMed

    Abreu, Murilo S; Giacomini, Ana C V V; Rodriguez, Rubens; Kalueff, Allan V; Barcellos, Leonardo J G

    2017-03-01

    Olfaction plays a key role in modulating behavioral and physiological responses of various animal species, including fishes. Olfactory deficits can be induced in fish experimentally, and utilized to examine the role of olfaction in their normal and pathological behaviors. Here, we examine whether experimental anosmia, evoked by ZnSO4 in adult zebrafish can be associated with behavioral and/or physiological responses. We show that experimental ZnSO4-induced anosmia caused acute, but not prolonged, anxiogenic-like effects on zebrafish behavior tested in the novel tank test. The procedure also elevated whole-body cortisol levels in zebrafish. Moreover, ZnSO4 treatment, but not sham, produced damage to olfactory epithelium, inducing overt basal cell vacuolization and intercellular edema. The loss of olfaction, assessed by the fish food preference behavior in the aquatic Y-maze, was present 1h, but not 24h, after the treatment. Collectively, this suggests that transient experimental anosmia by ZnSO4 modulates zebrafish behavior and olfaction, which can be used to evoke and assess their stress-related anxiety-like states.

  9. Using Zebrafish to Test the Genetic Basis of Human Craniofacial Diseases.

    PubMed

    Machado, R Grecco; Eames, B Frank

    2017-10-01

    Genome-wide association studies (GWASs) opened an innovative and productive avenue to investigate the molecular basis of human craniofacial disease. However, GWASs identify candidate genes only; they do not prove that any particular one is the functional villain underlying disease or just an unlucky genomic bystander. Genetic manipulation of animal models is the best approach to reveal which genetic loci identified from human GWASs are functionally related to specific diseases. The purpose of this review is to discuss the potential of zebrafish to resolve which candidate genetic loci are mechanistic drivers of craniofacial diseases. Many anatomic, embryonic, and genetic features of craniofacial development are conserved among zebrafish and mammals, making zebrafish a good model of craniofacial diseases. Also, the ability to manipulate gene function in zebrafish was greatly expanded over the past 20 y, enabling systems such as Gateway Tol2 and CRISPR-Cas9 to test gain- and loss-of-function alleles identified from human GWASs in coding and noncoding regions of DNA. With the optimization of genetic editing methods, large numbers of candidate genes can be efficiently interrogated. Finding the functional villains that underlie diseases will permit new treatments and prevention strategies and will increase understanding of how gene pathways operate during normal development.

  10. A Zebrafish Model of Myelodysplastic Syndrome Produced through tet2 Genomic Editing

    PubMed Central

    Gjini, Evisa; Mansour, Marc R.; Sander, Jeffry D.; Moritz, Nadine; Nguyen, Ashley T.; Kesarsing, Michiel; Gans, Emma; He, Shuning; Chen, Si; Ko, Myunggon; Kuang, You-Yi; Yang, Song; Zhou, Yi; Rodig, Scott; Zon, Leonard I.; Joung, J. Keith; Rao, Anjana

    2014-01-01

    The ten-eleven translocation 2 gene (TET2) encodes a member of the TET family of DNA methylcytosine oxidases that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) to initiate the demethylation of DNA within genomic CpG islands. Somatic loss-of-function mutations of TET2 are frequently observed in human myelodysplastic syndrome (MDS), which is a clonal malignancy characterized by dysplastic changes of developing blood cell progenitors, leading to ineffective hematopoiesis. We used genome-editing technology to disrupt the zebrafish Tet2 catalytic domain. tet2m/m (homozygous for the mutation) zebrafish exhibited normal embryonic and larval hematopoiesis but developed progressive clonal myelodysplasia as they aged, culminating in myelodysplastic syndromes (MDS) at 24 months of age, with dysplasia of myeloid progenitor cells and anemia with abnormal circulating erythrocytes. The resultant tet2m/m mutant zebrafish lines show decreased levels of 5hmC in hematopoietic cells of the kidney marrow but not in other cell types, most likely reflecting the ability of other Tet family members to provide this enzymatic activity in nonhematopoietic tissues but not in hematopoietic cells. tet2m/m zebrafish are viable and fertile, providing an ideal model to dissect altered pathways in hematopoietic cells and, for small-molecule screens in embryos, to identify compounds with specific activity against tet2 mutant cells. PMID:25512612

  11. Identification of Modulators of Hair Cell Regeneratin in the Zebrafish Lateral Line

    PubMed Central

    Namdaran, Parhum; Reinhart, Katherine E.; Owens, Kelly N.; Raible, David W.; Rubel, Edwin W

    2012-01-01

    The external location of the zebrafish lateral line makes it a powerful model for studying mechanosensory hair cell regeneration. We have developed a chemical screen to identify FDA-approved drugs and biologically active compounds that modulate hair cell regeneration in zebrafish. Of the 1,680 compounds evaluated, we identified 2 enhancers and 6 inhibitors of regeneration. The two enhancers, dexamethasone and prednisolone, are synthetic glucocorticoids that potentiated hair cell numbers during regeneration and also induced hair cell addition in the absence of damage. BrdU analysis confirmed that the extra hair cells arose from mitotic activity. We found that dexamethasone and prednisolone, like other glucocorticoids, suppress zebrafish caudal fin regeneration, indicating that hair cell regeneration occurs by a distinctly different process. Further analyses of the regeneration inhibitors revealed that two of the six, flubendazole and topotecan, significantly suppress hair cell regeneration by preventing proliferation of hair cell precursors. Flubendazole halted support cell division in M-phase, possibly by interfering with normal microtubule activity. Topotecan, a topoisomerase inhibitor, killed both hair cells and proliferating hair cell precursors. A third inhibitor, fulvestrant, moderately delays hair cell regeneration by reducing support cell proliferation. Our observation that hair cells do not regenerate when support cell proliferation is impeded confirms previous observations that cell division is the primary route for hair cell regeneration after neomycin treatment in zebrafish. PMID:22399774

  12. Influences of textured substrates on the heart rate of developing zebrafish embryos.

    PubMed

    Chen, Chia-Yun; Chen, Chia-Yuan

    2013-07-05

    Identification of the effects of different textured substrates on zebrafish (Danio rerio) embryos provides insights into the influence of external stimuli on normal cardiovascular functions in the developmental stages of the embryos. This knowledge can be used in numerous genetic studies using zebrafish as an animal model as well as in bioanalytical assays using digital microfluidics. In this study, zebrafish embryos were systematically positioned and in vivo imaged on four types of silicon substrates. These substrates exhibited surface textures and surface wettability that were well modulated by wet chemical etching. The heart rate of the developing embryos significantly increased by 9.1% upon exposure to textured Si substrates with nanostructured surfaces compared with bare Si substrates. Modulation of surface wettability in the tested substrates also responded to the increase in the heart rate of the embryo; however, the effect of surface wettability on heart rate was slight compared with the effect of texture. In-depth experimental and statistical investigations of heart rate under the effects of substrate textures imply a pathway through which the inner mass of the embryo reacts to external stimuli. These findings contribute to zebrafish-related studies and suggest other factors to consider in the design of nanostructure-based microfluidics and other biomedical devices.

  13. Comparison of proteomic profiles in the zebrafish retina during experimental degeneration and regeneration

    PubMed Central

    Eastlake, Karen; Heywood, Wendy E.; Tracey-White, Dhani; Aquino, Erika; Bliss, Emily; Vasta, Gerardo R.; Mills, Kevin; Khaw, Peng T.; Moosajee, Mariya; Limb, G. Astrid

    2017-01-01

    Zebrafish spontaneously regenerate the retina after injury. Although the gene expression profile has been extensively studied in this species during regeneration, this does not reflect protein function. To further understand the regenerative process in the zebrafish, we compared the proteomic profile of the retina during injury and upon regeneration. Using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics (quadrupole time of flight LC-MS/MS), we analysed the retina of adult longfin wildtype zebrafish at 0, 3 and 18 days after Ouabain injection. Gene ontology analysis indicates reduced metabolic processing, and increase in fibrin clot formation, with significant upregulation of fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1, and vitellogenin-6 during degeneration when compared to normal retina. In addition, cytoskeleton and membrane transport proteins were considerably altered during regeneration, with the highest fold upregulation observed for tubulin beta 2 A, histone H2B and brain type fatty acid binding protein. Key proteins identified in this study may play an important role in the regeneration of the zebrafish retina and investigations on the potential regulation of these proteins may lead to the design of protocols to promote endogenous regeneration of the mammalian retina following retinal degenerative disease. PMID:28300160

  14. An assay for permeability of the zebrafish embryonic neuroepithelium.

    PubMed

    Chang, Jessica T; Sive, Hazel

    2012-10-24

    The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) (1,2). CSF is a protein rich fluid that is essential for normal brain development and function(3-6). In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes (7,8), while claudin 5a is necessary for tight junction formation (9). Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation. To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab(10), to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2). This dye retention assay can be used to analyze neuroepithelial permeability in a

  15. Three-dimensional reconstruction and measurements of zebrafish larvae from high-throughput axial-view in vivo imaging

    PubMed Central

    Guo, Yuanhao; Veneman, Wouter J.; Spaink, Herman P.; Verbeek, Fons J.

    2017-01-01

    High-throughput imaging is applied to provide observations for accurate statements on phenomena in biology and this has been successfully applied in the domain of cells, i.e. cytomics. In the domain of whole organisms, we need to take the hurdles to ensure that the imaging can be accomplished with a sufficient throughput and reproducibility. For vertebrate biology, zebrafish is a popular model system for High-throughput applications. The development of the Vertebrate Automated Screening Technology (VAST BioImager), a microscope mounted system, enables the application of zebrafish high-throughput screening. The VAST BioImager contains a capillary that holds a zebrafish for imaging. Through the rotation of the capillary, multiple axial-views of a specimen can be acquired. For the VAST BioImager, fluorescence and/or confocal microscopes are used. Quantitation of a specific signal as derived from a label in one fluorescent channel requires insight in the zebrafish volume to be able to normalize quantitation to volume units. However, from the setup of the VAST BioImager, a specimen volume cannot be straightforwardly derived. We present a high-throughput axial-view imaging architecture based on the VAST BioImager. We propose profile-based 3D reconstruction to produce 3D volumetric representations for zebrafish larvae using the axial-views. Volume and surface area can then be derived from the 3D reconstruction to obtain the shape characteristics in high-throughput measurements. In addition, we develop a calibration and a validation of our methodology. From our measurements we show that with a limited amount of views, accurate measurements of volume and surface area for zebrafish larvae can be obtained. We have applied the proposed method on a range of developmental stages in zebrafish and produced metrical references for the volume and surface area for each stage. PMID:28663894

  16. Selenoprotein N is required for ryanodine receptor calcium release channel activity in human and zebrafish muscle.

    PubMed

    Jurynec, Michael J; Xia, Ruohong; Mackrill, John J; Gunther, Derrick; Crawford, Thomas; Flanigan, Kevin M; Abramson, Jonathan J; Howard, Michael T; Grunwald, David Jonah

    2008-08-26

    Mutations affecting the seemingly unrelated gene products, SepN1, a selenoprotein of unknown function, and RyR1, the major component of the ryanodine receptor intracellular calcium release channel, result in an overlapping spectrum of congenital myopathies. To identify the immediate developmental and molecular roles of SepN and RyR in vivo, loss-of-function effects were analyzed in the zebrafish embryo. These studies demonstrate the two proteins are required for the same cellular differentiation events and are needed for normal calcium fluxes in the embryo. SepN is physically associated with RyRs and functions as a modifier of the RyR channel. In the absence of SepN, ryanodine receptors from zebrafish embryos or human diseased muscle have altered biochemical properties and have lost their normal sensitivity to redox conditions, which likely accounts for why mutations affecting either factor lead to similar diseases.

  17. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    PubMed

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  18. N-cadherin is required for cytodifferentiation during zebrafish odontogenesis.

    PubMed

    Verstraeten, B; van Hengel, J; Sanders, E; Van Roy, F; Huysseune, A

    2013-04-01

    N-cadherin is a well-studied classic cadherin involved in multiple developmental processes and is also known to have a signaling function. Using the zebrafish (Danio rerio) as a model, we tested the hypothesis that tooth morphogenesis is accompanied by dynamic changes in N-cadherin distribution and that absence of N-cadherin disturbs tooth development. N-cadherin, encoded by the gene cdh2, is absent during the initiation and morphogenesis stages of both primary (first-generation) and replacement teeth, as demonstrated by immunohistochemistry. However, N-cadherin is up-regulated at the onset of differentiation of cells of the inner dental epithelium and the dental papilla, i.e., the ameloblasts and odontoblasts, respectively. In the inner dental epithelium, N-cadherin is co-expressed with E-cadherin, excluding the occurrence of cadherin switching such as observed during human tooth development. While early lethality of N-cadherin knockout mice prevents any functional study of N-cadherin in mouse odontogenesis, zebrafish parachute (pac) mutants, deficient for N-cadherin, survive beyond the age when primary teeth normally start to form. In these mutants, the first tooth forms, but its development stops at the early cytodifferentiation stage. N-cadherin deficiency also completely inhibits the development of the other first-generation teeth, possibly due to the absence of N-cadherin signaling once the first tooth has differentiated.

  19. Locomotor development of zebrafish (Danio rerio) under novel hydrodynamic conditions.

    PubMed

    Danos, Nicole

    2012-02-01

    The kinematics, neuromuscular control, and hydrodynamic aspects of normal locomotor activity in larval zebrafish have been extensively studied. Although locomotion depends heavily on the fluid properties of water, we have little knowledge of what sensory and developmental cues zebrafish larvae receive from their interaction with the fluid medium in which they grow. In this study, I manipulate the viscosity of water in which larvae grow until 5 and 7 days postfertilization (dpf) and record the kinematics of routine turns in their growth medium. Larvae are then transferred to a new medium of different viscosity and filmed again after short and long acclimation periods. Four hypotheses are tested: (1) larval kinematics are constrained by muscle activation patterns, (2) larval kinematics are guided by kinematic objectives, (3) routine turning control is independent of early locomotor experience, and (4) response to novel fluid environment is independent of developmental stage. The results indicate that a kinematic parameter, stage 1 angle, correlates with the kinematics of stage 1 while muscle activation patterns likely constrain stage 2. Development of this behavior is not dependent on locomotor experience both at 5 and 7 dpf, although the two age groups respond differently to increased viscosity. © 2012 WILEY PERIODICALS, INC.

  20. Methods for generating and colonizing gnotobiotic zebrafish

    PubMed Central

    Pham, Linh N.; Kanther, Michelle; Semova, Ivana; Rawls, John F.

    2008-01-01

    Vertebrates are colonized at birth by complex and dynamic communities of microorganisms that can contribute significantly to host health and disease. The ability to raise animals in the absence of microorganisms has been a powerful tool for elucidating the relationships between animal hosts and their microbial residents. The optical transparency of the developing zebrafish and relative ease of generating germ-free zebrafish makes it an attractive model organism for gnotobiotic research. Here we provide a protocol for: generating zebrafish embryos; deriving and rearing germ-free zebrafish; and colonizing zebrafish with microorganisms. Using these methods, we typically obtain 80–90% sterility rates in our germ-free derivations with 90% survival in germ-free animals and 50–90% survival in colonized animals through larval stages. Obtaining embryos for derivation requires approximately 1–2 hours with a 3–8 hour incubation period prior to derivation. Derivation of germ-free animals takes 1–1.5 hours, and daily maintenance requires 1–2 hours. PMID:19008873

  1. Regeneration of the Pancreas in Adult Zebrafish

    PubMed Central

    Moss, Jennifer B.; Koustubhan, Punita; Greenman, Melanie; Parsons, Michael J.; Walter, Ingrid; Moss, Larry G.

    2009-01-01

    OBJECTIVE Regenerating organs in diverse biological systems have provided clues to processes that can be harnessed to repair damaged tissue. Adult mammalian β-cells have a limited capacity to regenerate, resulting in diabetes and lifelong reliance on insulin. Zebrafish have been used as a model for the regeneration of many organs. We demonstrate the regeneration of adult zebrafish pancreatic β-cells. This nonmammalian model can be used to define pathways for islet-cell regeneration in humans. RESEARCH DESIGN AND METHODS Adult transgenic zebrafish were injected with a single high dose of streptozotocin or metronidazole and anesthetized at 3, 7, or 14 days or pancreatectomized. Blood glucose measurements were determined and gut sections were analyzed using specific endocrine, exocrine, and duct cell markers as well as markers for dividing cells. RESULTS Zebrafish recovered rapidly without the need for insulin injections, and normoglycemia was attained within 2 weeks. Although few proliferating cells were present in vehicles, ablation caused islet destruction and a striking increase of proliferating cells, some of which were Pdx1 positive. Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively β-cells. CONCLUSIONS The ability of the zebrafish to regenerate a functional pancreas using chemical, genetic, and surgical approaches enabled us to identify patterns of cell proliferation in islets and ducts. Further study of the origin and contribution of proliferating cells in reestablishing islet function could provide strategies for treating human diseases. PMID:19491207

  2. Latent learning in zebrafish (Danio rerio).

    PubMed

    Gómez-Laplaza, Luis M; Gerlai, Robert

    2010-04-02

    The zebrafish may represent an excellent compromise between system complexity and practical simplicity for behavioral brain research. It may be particularly appropriate for large scale screening studies whose aim is to identify mutants with altered phenotypes or novel compounds with particular efficacy. For example, the zebrafish may have utility in the analysis of the biological mechanisms of learning and memory. Although learning and memory have been extensively studied and hundreds of underlying molecular mechanisms have been identified, this number may represent only the fraction of genes involved in these complex brain functions. Thus large scale mutagenesis screens may have utility. In order for such screens to succeed, appropriate screening paradigms must be developed. The first step in this research is the characterization of learning and memory capabilities of zebrafish and the development of automatable tasks. Here we show that zebrafish is capable of latent learning, i.e. can acquire memory of their environment after being allowed to explore it. For example, we found experimental zebrafish that experienced an open left tunnel or an open right tunnel of a maze during the unrewarded exploration phase of the test to show the appropriate side bias during a probe trial when they had to swim to a group of conspecifics (the reward). Given that exploration of the maze does not require the presence of the experimenter and the probe trial, during which the subjects are video-recorded and their memory is tested, is short, we argue that the paradigm has utility in high-throughput screening.

  3. Production of Androgenetic Zebrafish (Danio Rerio)

    PubMed Central

    Corley-Smith, G. E.; Lim, C. J.; Brandhorst, B. P.

    1996-01-01

    To help investigate the evolutionary origin of the imprinting (parent-of-origin mono-allelic expression) of paternal genes observed in mammals, we constructed haploid and diploid androgenetic zebrafish (Danio rerio). Haploid androgenotes were produced by fertilizing eggs that had been X-ray irradiated to eliminate the maternal genome. Subsequent inhibition of the first mitotic division of haploid androgenotes by heat shock produced diploid androgenotes. The lack of inheritance of maternal-specific DNA markers (RAPD and SSR) by putative diploid and haploid androgenotes confirmed the androgenetic origin of their genomes. Marker analysis was performed on 18 putative androgenotes (five diploids and 13 haploids) from six families. None of 157 maternal-specific RAPD markers analyzed, some of which were apparently homozygous, were passed on to any of these putative androgenotes. A mean of 7.7 maternal-specific markers were assessed per family. The survival of androgenetic zebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development. Production of haploid androgenotes can be used to determine the meiotic recombination rate in male zebrafish. Androgenesis may also provide useful information about the mechanism of sex determination in zebrafish. PMID:8846903

  4. Transcriptome Analysis of Zebrafish Embryogenesis Using Microarrays

    PubMed Central

    Mathavan, Sinnakaruppan; Lee, Serene G. P; Mak, Alicia; Miller, Lance D; Murthy, Karuturi Radha Krishna; Govindarajan, Kunde R; Tong, Yan; Wu, Yi Lian; Lam, Siew Hong; Yang, Henry; Ruan, Yijun; Korzh, Vladimir; Gong, Zhiyuan; Liu, Edison T; Lufkin, Thomas

    2005-01-01

    Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html). PMID:16132083

  5. Defects of the Glycinergic Synapse in Zebrafish.

    PubMed

    Ogino, Kazutoyo; Hirata, Hiromi

    2016-01-01

    Glycine mediates fast inhibitory synaptic transmission. Physiological importance of the glycinergic synapse is well established in the brainstem and the spinal cord. In humans, the loss of glycinergic function in the spinal cord and brainstem leads to hyperekplexia, which is characterized by an excess startle reflex to sudden acoustic or tactile stimulation. In addition, glycinergic synapses in this region are also involved in the regulation of respiration and locomotion, and in the nociceptive processing. The importance of the glycinergic synapse is conserved across vertebrate species. A teleost fish, the zebrafish, offers several advantages as a vertebrate model for research of glycinergic synapse. Mutagenesis screens in zebrafish have isolated two motor defective mutants that have pathogenic mutations in glycinergic synaptic transmission: bandoneon (beo) and shocked (sho). Beo mutants have a loss-of-function mutation of glycine receptor (GlyR) β-subunit b, alternatively, sho mutant is a glycinergic transporter 1 (GlyT1) defective mutant. These mutants are useful animal models for understanding of glycinergic synaptic transmission and for identification of novel therapeutic agents for human diseases arising from defect in glycinergic transmission, such as hyperekplexia or glycine encephalopathy. Recent advances in techniques for genome editing and for imaging and manipulating of a molecule or a physiological process make zebrafish more attractive model. In this review, we describe the glycinergic defective zebrafish mutants and the technical advances in both forward and reverse genetic approaches as well as in vivo visualization and manipulation approaches for the study of the glycinergic synapse in zebrafish.

  6. Zebrafish as an emerging model for studying complex brain disorders

    PubMed Central

    Kalueff, Allan V.; Stewart, Adam Michael; Gerlai, Robert

    2014-01-01

    The zebrafish (Danio rerio) is rapidly becoming a popular model organism in pharmacogenetics and neuropharmacology. Both larval and adult zebrafish are currently used to increase our understanding of brain function, dysfunction, and their genetic and pharmacological modulation. Here we review the developing utility of zebrafish in the analysis of complex brain disorders (including, for example, depression, autism, psychoses, drug abuse and cognitive disorders), also covering zebrafish applications towards the goal of modeling major human neuropsychiatric and drug-induced syndromes. We argue that zebrafish models of complex brain disorders and drug-induced conditions have become a rapidly emerging critical field in translational neuropharmacology research. PMID:24412421

  7. Visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy.

    PubMed

    Gfrerer, Lisa; Dougherty, Max; Liao, Eric C

    2013-09-30

    Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

  8. Comparative effects of nodularin and microcystin-LR in zebrafish: 2. Uptake and molecular effects in eleuthero-embryos and adult liver with focus on endoplasmic reticulum stress.

    PubMed

    Faltermann, Susanne; Grundler, Verena; Gademann, Karl; Pernthaler, Jakob; Fent, Karl

    2016-02-01

    Microcystin (MC) and nodularin are structurally similar cyanobacterial toxins that inhibit protein phosphatases. Additional modes of action are poorly known, in particular for nodularin. In our associated work, we showed that active cellular uptake is mediated by the organic anion transporting polypeptide drOatp1d1 in zebrafish (Faltermann et al., 2016). Here, we assessed the transcriptional expression of three genes encoding three uptake transporters during embryonic development from 24h post fertilization (hpf) to 168 hpf. Transcripts of drOatp1d1 and drOatp2b1 are present at 24 hpf. The abundance increased after hatching and remained about constant up to 168 hpf. Transcripts of drOatp2b1 were most abundant, while drOapt1f transcripts showed very low relative abundance compared to drOatp1d1 and drOatp2b1. We further demonstrated the uptake of fluorescent labeled MC-LR in eleuthero-embryos and its accumulation in the glomerulus of the pronephros. An important molecular effect of MC-LR in human liver cells is the induction of endoplasmic reticulum (ER)-stress. Here, we investigated, whether MC-LR and nodularin similarly lead to induction of ER-stress in zebrafish by analyzing changes of mRNA levels of genes indicative of ER-stress. In zebrafish liver organ cultures short- and long-term exposures to 0.15 and 0.3 μmol L(-1) MC-LR, and 0.5 and 1 μM L(-1) nodularin led to significant transcriptional induction of several ER-stress marker genes, including the chaperone glucose regulated protein 78 (bip), the spliced form of x-box binding protein (xbp-1s), the CCAAT-enhancer-binding protein homologous protein (chop) and activating transcription factor 4 (atf4). Furthermore, strong transcriptional changes occurred for tumor necrosis factor alpha (tnfa) and dual specificity phosphatase 5 (dusp5), associated with mitogen activated protein kinase (MAPK) pathway. However, no alterations in transcript levels of pro-apoptotic genes Bcl-2 like protein 4 (bax) and p53 occurred

  9. Zebrafish models of dyslipidemia: Relevance to atherosclerosis and angiogenesis

    PubMed Central

    Fang, Longhou; Liu, Chao; Miller, Yury I.

    2013-01-01

    Lipid and lipoprotein metabolism in zebrafish and in humans are remarkably similar. Zebrafish express all major nuclear receptors, lipid transporters, apolipoproteins and enzymes involved in lipoprotein metabolism. Unlike mice, zebrafish express cetp and the Cetp activity is detected in zebrafish plasma. Feeding zebrafish a high cholesterol diet, without any genetic intervention, results in significant hypercholesterolemia and robust lipoprotein oxidation, making zebrafish an attractive animal model to study mechanisms relevant to early development of human atherosclerosis. These studies are facilitated by the optical transparency of zebrafish larvae and the availability of transgenic zebrafish expressing fluorescent proteins in endothelial cells and macrophages. Thus, vascular processes can be monitored in live animals. In this review article we discuss recent advances in using dyslipidemic zebrafish in atherosclerosis-related studies. We also summarize recent work connecting lipid metabolism with regulation of angiogenesis, the work that considerably benefited from using the zebrafish model. These studies uncovered the role of aibp, abca1, abcg1, mtp, apoB and apoC2 in regulation of angiogenesis in zebrafish and paved the way for future studies in mammals, which may suggest new therapeutic approaches to modulation of excessive or diminished angiogenesis contributing to the pathogenesis of human disease. PMID:24095954

  10. New tides: using zebrafish to study renal regeneration.

    PubMed

    McCampbell, Kristen K; Wingert, Rebecca A

    2014-02-01

    Over the past several decades, the zebrafish has become one of the major vertebrate model organisms used in biomedical research. In this arena, the zebrafish has emerged as an applicable system for the study of kidney diseases and renal regeneration. The relevance of the zebrafish model for nephrology research has been increasingly appreciated as the understanding of zebrafish kidney structure, ontogeny, and the response to damage has steadily expanded. Recent studies have documented the amazing regenerative characteristics of the zebrafish kidney, which include the ability to replace epithelial populations after acute injury and to grow new renal functional units, termed nephrons. Here we discuss how nephron composition is conserved between zebrafish and mammals, and highlight how recent findings from zebrafish studies utilizing transgenic technologies and chemical genetics can complement traditional murine approaches in the effort to dissect how the kidney responds to acute damage and identify therapeutics that enhance human renal regeneration. Copyright © 2014 Mosby, Inc. All rights reserved.

  11. Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

    PubMed Central

    Lopez-Ramirez, Miguel A.; Calvo, Charles-Félix; Ristori, Emma; Thomas, Jean-Léon; Nicoli, Stefania

    2016-01-01

    The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish. PMID:26967835

  12. Developing 'integrative' zebrafish models of behavioral and metabolic disorders.

    PubMed

    Nguyen, Michael; Yang, Ester; Neelkantan, Nikhil; Mikhaylova, Alina; Arnold, Raymond; Poudel, Manoj K; Stewart, Adam Michael; Kalueff, Allan V

    2013-11-01

    Recently, the pathophysiological overlap between metabolic and mental disorders has received increased recognition. Zebrafish (Danio rerio) are rapidly becoming a popular model organism for translational biomedical research due to their genetic tractability, low cost, quick reproductive cycle, and ease of behavioral, pharmacological or genetic manipulation. High homology to mammalian physiology and the availability of well-developed assays also make the zebrafish an attractive organism for studying human disorders. Zebrafish neurobehavioral and endocrine phenotypes show promise for the use of zebrafish in studies of stress, obesity and related behavioral and metabolic disorders. Here, we discuss the parallels between zebrafish and other model species in stress and obesity physiology, as well as outline the available zebrafish models of weight gain, metabolic deficits, feeding, stress, anxiety and related behavioral disorders. Overall, zebrafish demonstrate a strong potential for modeling human behavioral and metabolic disorders, and their comorbidity.

  13. European Zebrafish Meeting 2015 Husbandry Session Report.

    PubMed

    Varga, Zoltán M; Wilson, Carole; Alestrøm, Peter

    2016-06-01

    A workshop to address husbandry and animal welfare was held during the 9th European Zebrafish Meeting in Oslo, Norway, from June 28 to July 2, 2015. The husbandry workshop took place on Monday, June 29, and was well attended by ∼100 audience members. It highlighted problems arising from the diversity of current husbandry practices and included presentations on recent efforts to find common husbandry and animal welfare standards from a variety of international contributors, from Norway, Portugal, the United Kingdom, as well as the United States and Japan. Presentations included zebrafish and medaka as representatives of aquatic species used in biomedical research and addressed a diverse range of topics such as proposed European guidelines for zebrafish husbandry, general fish facility health and husbandry standards, cryopreservation, publication standards, and feeding strategies. The workshop highlighted the desire to develop common husbandry standards for the aquatic research community across the world.

  14. Conditional gene-trap mutagenesis in zebrafish.

    PubMed

    Maddison, Lisette A; Li, Mingyu; Chen, Wenbiao

    2014-01-01

    Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene-trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial- and temporal-specific gene inactivation but also permit spatial- and temporal-specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.

  15. 15 years of zebrafish chemical screening

    PubMed Central

    Rennekamp, Andrew J.; Peterson, Randall T.

    2015-01-01

    In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies. PMID:25461724

  16. In vitro development of zebrafish vascular networks.

    PubMed

    Ibrahim, Muhammad; Richardson, Michael K

    2017-02-09

    A major limitation to culturing tissues and organs is the lack of a functional vascular network in vitro. The zebrafish possess many useful properties which makes it a promising model for such studies. Unfortunately, methods of culturing endothelial cells from this species are not well characterised. Here, we tried two methods (embryoid body culture and organ explants from transgenic zebrafish kdrl:GFP embryos) to develop in vitro vascular networks. In the kdrl:GFP line, endothelial cells expresses green fluorescent protein, which allows to track the vascular development in live cultures. We found that embryoid bodies showed significantly longer and wider branches of connected endothelial cells when grown in a microfluidic system than in static culture. Similarly, sprouting of kdrl:GFP(+) cells from the tissue explants was observed in a 3D hydrogel matrix. This study is a step towards the development of zebrafish vascular networks in vitro.

  17. 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure Prevents Cardiac Valve Formation in Developing Zebrafish

    PubMed Central

    Mehta, Vatsal; Peterson, Richard E.; Heideman, Warren

    2008-01-01

    Cardiovascular malformations are one of the most common congenital birth defects observed in humans. Defects in cardiac valves disrupt normal blood flow. Zebrafish are an outstanding experimental model for studying the effects that environmental contaminants have on developmental processes. Previous research has shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes blood regurgitation in the heart and reduces peripheral blood flow in embryonic zebrafish, suggesting some form of valve failure. To test this we used video microscopy to examine valve function and structure in developing zebrafish exposed to TCDD. TCDD exposure produced blood regurgitation at both the atrioventricular (AV) and bulboventricular (BV) junctions. In marked contrast to control embryos exposed to the vehicle dimethyl sulfoxide, embryos exposed to TCDD failed to form valve leaflets as the heart matured. In addition, whereas TCDD did not block initial formation of the bulbus arteriosus, we found that TCDD exposure prevented the normal growth and development of this portion of the outflow tract. TCDD altered the localization of endothelial cells at the AV and BV junctions and altered the localized expression of mRNAs bmp4 and notch1b normally associated with the nascent valves. Taken together, our results demonstrate that although TCDD does not prevent the initial specification of the presumptive valve locations, TCDD exposure produces severe alterations in valve development, leading to blood regurgitation and failing circulation in the developing zebrafish. PMID:18477685

  18. 2,3,7,8-Tetrachlorodibenzo-p-dioxin exposure prevents cardiac valve formation in developing zebrafish.

    PubMed

    Mehta, Vatsal; Peterson, Richard E; Heideman, Warren

    2008-08-01

    Cardiovascular malformations are one of the most common congenital birth defects observed in humans. Defects in cardiac valves disrupt normal blood flow. Zebrafish are an outstanding experimental model for studying the effects that environmental contaminants have on developmental processes. Previous research has shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes blood regurgitation in the heart and reduces peripheral blood flow in embryonic zebrafish, suggesting some form of valve failure. To test this we used video microscopy to examine valve function and structure in developing zebrafish exposed to TCDD. TCDD exposure produced blood regurgitation at both the atrioventricular (AV) and bulboventricular (BV) junctions. In marked contrast to control embryos exposed to the vehicle dimethyl sulfoxide, embryos exposed to TCDD failed to form valve leaflets as the heart matured. In addition, whereas TCDD did not block initial formation of the bulbus arteriosus, we found that TCDD exposure prevented the normal growth and development of this portion of the outflow tract. TCDD altered the localization of endothelial cells at the AV and BV junctions and altered the localized expression of mRNAs bmp4 and notch1b normally associated with the nascent valves. Taken together, our results demonstrate that although TCDD does not prevent the initial specification of the presumptive valve locations, TCDD exposure produces severe alterations in valve development, leading to blood regurgitation and failing circulation in the developing zebrafish.

  19. 3D Finite Element Electrical Model of Larval Zebrafish ECG Signals

    PubMed Central

    Crowcombe, James; Dhillon, Sundeep Singh; Hurst, Rhiannon Mary; Egginton, Stuart; Müller, Ferenc; Sík, Attila; Tarte, Edward

    2016-01-01

    Assessment of heart function in zebrafish larvae using electrocardiography (ECG) is a potentially useful tool in developing cardiac treatments and the assessment of drug therapies. In order to better understand how a measured ECG waveform is related to the structure of the heart, its position within the larva and the position of the electrodes, a 3D model of a 3 days post fertilisation (dpf) larval zebrafish was developed to simulate cardiac electrical activity and investigate the voltage distribution throughout the body. The geometry consisted of two main components; the zebrafish body was modelled as a homogeneous volume, while the heart was split into five distinct regions (sinoatrial region, atrial wall, atrioventricular band, ventricular wall and heart chambers). Similarly, the electrical model consisted of two parts with the body described by Laplace’s equation and the heart using a bidomain ionic model based upon the Fitzhugh-Nagumo equations. Each region of the heart was differentiated by action potential (AP) parameters and activation wave conduction velocities, which were fitted and scaled based on previously published experimental results. ECG measurements in vivo at different electrode recording positions were then compared to the model results. The model was able to simulate action potentials, wave propagation and all the major features (P wave, R wave, T wave) of the ECG, as well as polarity of the peaks observed at each position. This model was based upon our current understanding of the structure of the normal zebrafish larval heart. Further development would enable us to incorporate features associated with the diseased heart and hence assist in the interpretation of larval zebrafish ECGs in these conditions. PMID:27824910

  20. Teratological effects of a panel of sixty water-soluble toxicants on zebrafish development.

    PubMed

    Ali, Shaukat; Aalders, Jeffrey; Richardson, Michael K

    2014-04-01

    The zebrafish larva is a promising whole-animal model for safety pharmacology, environmental risk assessment, and developmental toxicity. This model has been used for the high-throughput toxicity screening of various compounds. Our aim here is to identify possible phenotypic markers of teratogenicity in zebrafish embryos that could be used for the assaying compounds for reproductive toxicity. We have screened a panel of 60 water-soluble toxicants to examine their effects on zebrafish development. A total of 22,080 wild-type zebrafish larvae were raised in 250 μL defined buffer in 96-well plates at a plating density of one embryo per well. They were exposed for a 96-h period starting at 24 h post-fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for developmental toxicity assessment. A total of 9017 survivors were analyzed at 5 days post-fertilization for nine phenotypes, namely, (1) normal, (2) pericardial oedema, (3) yolk sac oedema, (4) melanophores dispersed, (5) bent tail tip, (6) bent body axis, (7) abnormal Meckel's cartilage, (8) abnormal branchial arches, and (9) uninflated swim bladder. For each toxicant, the EC50 (concentration required to produce one or more of these abnormalities in 50% of embryos) was also calculated. For the majority of toxicants (55/60) there was, at the population level, a statistically significant, concentration-dependent increase in the incidence of abnormal phenotypes among survivors. The commonest abnormalities were pericardial oedema, yolk sac oedema, dispersed melanophores, and uninflated swim bladder. It is possible therefore that these could prove to be general indicators of reproductive toxicity in the zebrafish embryo assay.

  1. Teratological Effects of a Panel of Sixty Water-Soluble Toxicants on Zebrafish Development

    PubMed Central

    Ali, Shaukat; Aalders, Jeffrey

    2014-01-01

    Abstract The zebrafish larva is a promising whole-animal model for safety pharmacology, environmental risk assessment, and developmental toxicity. This model has been used for the high-throughput toxicity screening of various compounds. Our aim here is to identify possible phenotypic markers of teratogenicity in zebrafish embryos that could be used for the assaying compounds for reproductive toxicity. We have screened a panel of 60 water-soluble toxicants to examine their effects on zebrafish development. A total of 22,080 wild-type zebrafish larvae were raised in 250 μL defined buffer in 96-well plates at a plating density of one embryo per well. They were exposed for a 96-h period starting at 24 h post-fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for developmental toxicity assessment. A total of 9017 survivors were analyzed at 5 days post-fertilization for nine phenotypes, namely, (1) normal, (2) pericardial oedema, (3) yolk sac oedema, (4) melanophores dispersed, (5) bent tail tip, (6) bent body axis, (7) abnormal Meckel's cartilage, (8) abnormal branchial arches, and (9) uninflated swim bladder. For each toxicant, the EC50 (concentration required to produce one or more of these abnormalities in 50% of embryos) was also calculated. For the majority of toxicants (55/60) there was, at the population level, a statistically significant, concentration-dependent increase in the incidence of abnormal phenotypes among survivors. The commonest abnormalities were pericardial oedema, yolk sac oedema, dispersed melanophores, and uninflated swim bladder. It is possible therefore that these could prove to be general indicators of reproductive toxicity in the zebrafish embryo assay. PMID:24650241

  2. Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos

    SciTech Connect

    McAleer, Mary Frances; Duffy, Kevin T.; Davidson, William R.; Kari, Gabor; Dicker, Adam P.; Rodeck, Ulrich; Wickstrom, Eric . E-mail: eric@tesla.jci.tju.edu

    2006-10-01

    Purpose: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance Radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. Methods and Materials: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. Results: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the 'curly up' phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. Conclusions: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

  3. Hedgehog signaling is required at multiple stages of zebrafish tooth development

    PubMed Central

    2010-01-01

    Background The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. Results We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. Conclusion We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution. PMID:21118524

  4. The Zebrafish Ortholog of TRPV1 Is Required for Heat-Induced Locomotion

    PubMed Central

    Gau, Philia; Poon, Jason; Ufret-Vincenty, Carmen; Snelson, Corey D.; Gordon, Sharona E.; Raible, David W.

    2013-01-01

    The ability to detect hot temperatures is critical to maintaining body temperature and avoiding injury in diverse animals from insects to mammals. Zebrafish embryos, when given a choice, actively avoid hot temperatures and display an increase in locomotion similar to that seen when they are exposed to noxious compounds such as mustard oil. Phylogenetic analysis suggests that the single zebrafish ortholog of TRPV1/2 may have arisen from an evolutionary precursor of the mammalian TRPV1 and TRPV2. As opposed to TRPV2, mammalian TRPV1 is essential for environmentally relevant heat sensation. In the present study, we provide evidence that the zebrafish TRPV1 ion channel is also required for the sensation of heat. Contrary to development in mammals, zebrafish TRPV1+ neurons arise during the first wave of somatosensory neuron development, suggesting a vital importance of thermal sensation in early larval survival. In vitro analysis showed that zebrafish TRPV1 acts as a molecular sensor of environmental heat (≥25°C) that is distinctly lower than the sensitivity of the mammalian form (≥42°C) but consistent with thresholds measured in behavioral assays. Using in vivo calcium imaging with the genetically encoded calcium sensor GCaMP3, we show that TRPV1-expressing trigeminal neurons are activated by heat at behaviorally relevant temperatures. Using knock-down studies, we also show that TRPV1 is required for normal heat-induced locomotion. Our results demonstrate for the first time an ancient role for TRPV1 in the direct sensation of environmental heat and show that heat sensation is adapted to reflect species-dependent requirements in response to environmental stimuli. PMID:23516290

  5. 3D Finite Element Electrical Model of Larval Zebrafish ECG Signals.

    PubMed

    Crowcombe, James; Dhillon, Sundeep Singh; Hurst, Rhiannon Mary; Egginton, Stuart; Müller, Ferenc; Sík, Attila; Tarte, Edward

    2016-01-01

    Assessment of heart function in zebrafish larvae using electrocardiography (ECG) is a potentially useful tool in developing cardiac treatments and the assessment of drug therapies. In order to better understand how a measured ECG waveform is related to the structure of the heart, its position within the larva and the position of the electrodes, a 3D model of a 3 days post fertilisation (dpf) larval zebrafish was developed to simulate cardiac electrical activity and investigate the voltage distribution throughout the body. The geometry consisted of two main components; the zebrafish body was modelled as a homogeneous volume, while the heart was split into five distinct regions (sinoatrial region, atrial wall, atrioventricular band, ventricular wall and heart chambers). Similarly, the electrical model consisted of two parts with the body described by Laplace's equation and the heart using a bidomain ionic model based upon the Fitzhugh-Nagumo equations. Each region of the heart was differentiated by action potential (AP) parameters and activation wave conduction velocities, which were fitted and scaled based on previously published experimental results. ECG measurements in vivo at different electrode recording positions were then compared to the model results. The model was able to simulate action potentials, wave propagation and all the major features (P wave, R wave, T wave) of the ECG, as well as polarity of the peaks observed at each position. This model was based upon our current understanding of the structure of the normal zebrafish larval heart. Further development would enable us to incorporate features associated with the diseased heart and hence assist in the interpretation of larval zebrafish ECGs in these conditions.

  6. Hedgehog signaling is required at multiple stages of zebrafish tooth development.

    PubMed

    Jackman, William R; Yoo, James J; Stock, David W

    2010-11-30

    The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.

  7. Zebrafish (Danio rerio) fed vitamin E deficient diets produce embryos with increased morphologic abnormalities and mortality

    PubMed Central

    Miller, Galen W.; Labut, Edwin M.; Lebold, Katie M.; Floeter, Abby; Tanguay, Robert L.; Traber, Maret G.

    2011-01-01

    Vitamin E (α-tocopherol) is required to prevent fetal resorption in rodents. To study α–tocopherol’s role in fetal development, a non-placental model is required. Therefore, the zebrafish, an established developmental model organism, was studied by feeding the fish a defined diet with or without added α–tocopherol. Zebrafish (age: 4–6 w) were fed the deficient (E-), sufficient (E+), or lab diet up to 1 y. All groups showed similar growth rates. The exponential rate of α–tocopherol depletion up to ~80 day in E- zebrafish was 0.029 ± 0.006 nmol/g, equivalent to a depletion half-life of 25 ± 5 days. From age ~80 d, the E- fish (5 ± 3 nmol/g) contained ~50 times less α–tocopherol than the E+ or lab diet fish (369 ± 131 or 362 ± 107, respectively, P<0.05). E-depleted adults demonstrated decreased startle response suggesting neurologic deficits. Expression of selected oxidative stress and apoptosis genes from livers isolated from the zebrafish fed the three diets were evaluated by quantitative polymerase chain reaction and were not found to vary with vitamin E status. When E-depleted adults were spawned, they produced viable embryos with depleted α–tocopherol concentrations. The E- embryos exhibited a higher mortality (P<0.05) at 24 h post fertilization (hpf) and a higher combination of malformations and mortality (P<0.05) at 120 hpf than embryos from parents fed E+ or lab diets. This study documents for the first time that vitamin E is essential for normal zebrafish embryonic development. PMID:21684137

  8. [Construction and assessment of heart-specific green fluorescence zebrafish line].

    PubMed

    Peng, Xi-Yang; Chen, Ting-Fang; Huang, Ting; Jiang, Zhi-Gang; Wu, Xiu-Shan; Deng, Yun

    2013-04-01

    Using the promoter for cardiac myosin light chain 2 (cmlc2) gene, an expression vector pTol2-cmlc2-IRES- EGFP for making heart-specific expression of exogenous gene in transgenic zebrafish was generated previously. Here, we reported the construction of a transgenic zebrafish line which stably expresses EGFP using this vector, and the effects of EGFP on the heart development and cardiac function of this transgenic zebrafish line were preliminarily analyzed. The results showed that the green fluorescence signal of cmlc2:EGFP line under fluorescence microscopy specifically expressed in heart and faithfully recapitulated both the spatial and temporal expression patterns of endogenous cmlc2 gene revealed by in situ hybridization in the early developmental stages. The cardiac morphology and development of this transgenic zebrafish line remained to be normal. Furthermore, the heart morphology and physiological function of this transgenic line have been analyzed using M-mode analysis. The results showed that there was no significant difference between the cmlc2:EGFP and the wild type lines with respect to heart period, heart rate, diastolic surface area and systolic surface area, and fractional area change. No tachyarrhythmia was observed in the embryos from either line. Thus, the excessive expression of EGFP in this transgenic line seemed to exert no detrimental effects on the function and development of zebrafish hearts during early stages. Our study laid a foundation for the construction of exogenous gene transgenic line using pTol2-cmlc2-IRES-EGFP vector to study the function of genes that expressed in heart.

  9. Culturable Gut Microbiota Diversity in Zebrafish

    PubMed Central

    Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

    2012-01-01

    Abstract The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A–D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid

  10. Oxidative stress in zebrafish (Danio rerio) sperm.

    PubMed

    Hagedorn, Mary; McCarthy, Megan; Carter, Virginia L; Meyers, Stuart A

    2012-01-01

    Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely

  11. Irisin regulates cardiac physiology in zebrafish

    PubMed Central

    Sundarrajan, Lakshminarasimhan; Yeung, Chanel; Hahn, Logan; Weber, Lynn P.

    2017-01-01

    Irisin is a myokine encoded in its precursor fibronectin type III domain containing 5 (FNDC5). It is abundantly expressed in cardiac and skeletal muscle, and is secreted upon the activation of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1 alpha). We aimed to study the role of irisin on cardiac function and muscle protein regulation in zebrafish. Western blot analyses detected the presence of irisin protein (23 kDa) in zebrafish heart and skeletal muscle, and irisin immunoreactivity was detected in both tissues. Irisin siRNA treated samples did not show bands corresponding to irisin in zebrafish. In vitro studies found that treatment with irisin (0.1 nM) downregulated the expression of PGC-1 alpha, myostatin a, and b, while upregulating troponin C mRNA expression in zebrafish heart and skeletal muscle. Exogenous irisin (0.1 and 1 ng/g B.W) increased diastolic volume, heart rate and cardiac output, while knockdown of irisin (10 ng/g B.W) showed opposing effects on cardiovascular function. Irisin (1 and 10 ng/g B.W) downregulated PGC-1 alpha, myostatin a and b, and upregulated troponin C and troponin T2D mRNA expression. Meanwhile, knockdown of irisin showed opposing effects on troponin C, troponin T2D and myostatin a and b mRNAs in zebrafish heart and skeletal muscle. Collectively, these results identified muscle proteins as novel targets of irisin, and added irisin to the list of peptide modulators of cardiovascular physiology in zebrafish. PMID:28771499

  12. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

  13. Histone modifications in zebrafish development.

    PubMed

    Cunliffe, V T

    2016-01-01

    Reversible covalent histone modifications are known to influence spatiotemporal patterns of gene transcription during development. Here I review recent advances in the development and use of methods to analyze the distribution and functions of histone modifications in zebrafish chromatin. I discuss the roles of dynamic histone modification patterns at the promoters and enhancers of genes during the process of zygotic gene activation at blastula stages and the interplay between the molecular machinery responsible for histone modifications, chromatin remodeling and DNA methylation. Interactions are also described between developmentally regulated enhancer sequences and modified histones. A detailed method for chromatin immunoprecipitation using antibodies is provided, and I describe the use of high-throughput whole genome sequencing technology to generate DNA sequence data from chromatin immunoprecipitates. I also discuss computational approaches to integrating DNA sequence data obtained from chromatin immunoprecipitates with annotated reference genome sequences, transcriptome and methylome sequence data, transcription factor binding motif databases, and gene ontologies and describe the types of software tools currently available for visualizing the results. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Glyphosate induces neurotoxicity in zebrafish.

    PubMed

    Roy, Nicole M; Carneiro, Bruno; Ochs, Jeremy

    2016-03-01

    Glyphosate based herbicides (GBH) like Roundup(®) are used extensively in agriculture as well as in urban and rural settings as a broad spectrum herbicide. Its mechanism of action was thought to be specific only to plants and thus considered safe and non-toxic. However, mounting evidence suggests that GBHs may not be as safe as once thought as initial studies in frogs suggest that GBHs may be teratogenic. Here we utilize the zebrafish vertebrate model system to study early effects of glyphosate exposure using technical grade glyphosate and the Roundup(®) Classic formulation. We find morphological abnormalities including cephalic and eye reductions and a loss of delineated brain ventricles. Concomitant with structural changes in the developing brain, using in situ hybridization analysis, we detect decreases in genes expressed in the eye, fore and midbrain regions of the brain including pax2, pax6, otx2 and ephA4. However, we do not detect changes in hindbrain expression domains of ephA4 nor exclusive hindbrain markers krox-20 and hoxb1a. Additionally, using a Retinoic Acid (RA) mediated reporter transgenic, we detect no alterations in the RA expression domains in the hindbrain and spinal cord, but do detect a loss of expression in the retina. We conclude that glyphosate and the Roundup(®) formulation is developmentally toxic to the forebrain and midbrain but does not affect the hindbrain after 24 h exposure.

  15. Gene-specific differential response to anti-apoptotic therapies in zebrafish models of ocular coloboma

    PubMed Central

    Moosajee, Mariya; Shan, Xianghong; Gregory-Evans, Kevin

    2011-01-01

    Purpose We recently demonstrated that molecular therapy using aminoglycosides can overcome the underlying genetic defect in two zebrafish models of ocular coloboma and showed abnormal cell death to be a key feature associated with the optic fissure closure defects. In further studies to identify molecular therapies for this common congenital malformation, we now examine the effects of anti-apoptotic compounds in zebrafish models of ocular coloboma in vivo. Methods Two ocular coloboma zebrafish lines (pax2.1/noitu29a and lamb1/gupm189) were exposed to diferuloylmethane (curcumin) or benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD-fmk; a pan-caspase inhibitor) for up to 8 days post-fertilization. The effects of these compounds were assessed by morphology, histology, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and western blot analysis. Results The size of the coloboma in gup zebrafish mutants treated with diferuloylmethane was greatly reduced. In treated mutants a reduction in TUNEL staining and a 67% decrease in activated caspase-3 protein were observed. The release of cytochrome c from the mitochondria into the cytosol was reduced fourfold by in vivo diferuloylmethane treatment, suggesting that the drug was acting to inhibit the intrinsic apoptotic pathway. Inhibition of caspases directly with zVAD-fmk also resulted in a similar reduction in coloboma phenotype. Treatment with either diferuloylmethane or zVAD-fmk resulted in a statistically significant 1.4 fold increase in length of survival of these mutant zebrafish (p<0.001), which normally succumb to the lethal genetic mutation. In contrast, the coloboma phenotype in noi zebrafish mutants did not respond to either diferuloylmethane or zVAD-fmk exposure, even though inhibition of apoptotic cell death was observed by a reduction in TUNEL staining. Conclusions The differential sensitivity to anti-apoptotic agents in lamb1-deficient and pax2.1-deficient zebrafish models

  16. Dynamic focusing in the zebrafish beating heart

    NASA Astrophysics Data System (ADS)

    Andrés-Delgado, L.; Peralta, M.; Mercader, N.; Ripoll, J.

    2016-03-01

    Of the large amount of the animal models available for cardiac research, the zebrafish is extremely valuable due to its transparency during early stages of development. In this work a dual illumination laser sheet microscope with simultaneous dual camera imaging is used to image the beating heart at 200 fps, dynamically and selectively focusing inside the beating heart through the use of a tunable lens. This dual color dynamic focusing enables imaging with cellular resolution at unprecedented high frame rates, allowing 3D imaging of the whole beating heart of embryonic zebrafish.

  17. Zebrafish invade Valparaiso: third meeting and symposium of the Latin American zebrafish network.

    PubMed

    Whitlock, Kathleen E

    2014-12-01

    Zebrafish are an excellent model system for research and teaching. Because of their relatively low maintenance costs, beautiful and bountiful embryos, and tool box of molecular genetic technique, zebrafish are ideal for countries with smaller research budgets and less well-developed science infrastructure. For these reasons, zebrafish are growing in popularity as a model system for research in Latin America. In response to this growing need, we held the Third Latin American Zebrafish Network (LAZEN) Course and Symposium in Valparaiso, Chile, in April 4-13, 2014. The course covered a wide variety of topics from fish husbandry to outreach and ended with a symposium hosting excellent scientists from Latin America and beyond.

  18. Characterization and toxicology evaluation of chitosan nanoparticles on the embryonic development of zebrafish, Danio rerio.

    PubMed

    Wang, Yanbo; Zhou, Jinru; Liu, Lin; Huang, Changjiang; Zhou, Deqing; Fu, Linglin

    2016-05-05

    In the present study, chitosan nanoparticles were prepared, characterized and used to evaluate the embryonic toxicology on zebrafish (Danio rerio). The average particle size of chitosan nanoparticles was 84.86nm. The increased mortality and decreased hatching rate was found in the zebrafish embryo exposure to normal chitosan particles and chitosan nanoparticles with the increased addition concentration. At 120h post-fertilization (hpf), the rate of mortality were 25.0 and 44.4% in the groups treated with chitosan nanoparticles and normal chitosan particles at 250mg/L, respectively. At 72hpf, the hatching rate in the groups treated with normal chitosan particles were lower (P<0.01) at 300 and 400mg/L than those of the corresponding control groups, respectively. However, there were no significant differences between the groups treated with chitosan nanoparticles and the control groups across all the addition concentrations. More abundant typical malformation of embryos was observed in the groups treated with normal chitosan particles compared with those treated with chitosan nanoparticles. The LC50 (medium lethal concentration) of chitosan nanoparticles was 280mg/L at 96hpf and 270mg/L at 120hpf. As for normal chitosan particles, the LC50 was 257mg/L at both 96hpf and 120hpf. The TC50 (medium teratogenic concentration) of the zebrafish treated with chitosan nanoparticles and normal chitosan particles were 257mg/L and 137mg/L, respectively. It indicated that the chitosan nanoparticles were relatively more secure compared with normal chitosan particles.

  19. Eliminating zebrafish pbx proteins reveals a hindbrain ground state.

    PubMed

    Waskiewicz, Andrew Jan; Rikhof, Holly A; Moens, Cecilia B

    2002-11-01

    The vertebrate hindbrain is divided into serially homologous segments, the rhombomeres (r). Pbx and Hox proteins are hypothesized to form heterodimeric, DNA binding transcription complexes which specify rhombomere identities. Here, we show that eliminating zebrafish Lzr/Pbx4 and Pbx2 function prevents hindbrain segmentation and causes a wholesale anterior homeotic transformation of r2-r6, to r1 identity. We demonstrate that Pbx proteins interact with Hox paralog group 1 proteins to specify segment identities broadly within the hindbrain, and that this process involves the Pbx:Hox-1-dependent induction of Fgf signals in r4. We propose that in the absence of Pbx function, r2-r6 acquire a homogeneous ground state identity, that of r1, and that Pbx proteins, functioning primarily with their Hox partners, function to modify this ground state identity during normal hindbrain development.

  20. Functional bone histology of zebrafish reveals two types of endochondral ossification, different types of osteoblast clusters and a new bone type.

    PubMed

    Weigele, Jochen; Franz-Odendaal, Tamara A

    2016-07-01

    The zebrafish is as an important vertebrate animal model system for studying developmental processes, gene functions and signalling pathways. It is also used as a model system for the understanding of human developmental diseases including those related to the skeleton. However, surprisingly little is known about normal zebrafish skeletogenesis and osteogenesis. As in most vertebrates, it is commonly known that the bones of adult zebrafish are cellular unlike that of some other teleosts. After careful histological analyses of each zebrafish adult bone, we identified several acellular bones, with no entrapped osteocytes in addition to several cellular bones. We show that both cellular and acellular bones can even occur within the same skeletal element and transitions between these two cell types can be found. Furthermore, we describe two types of osteoblast clusters during skeletogenesis and two different types of endochondral ossification. The epiphyseal plate, for example, lacks a zone of calcification and a degradation zone with osteoblasts. A new bone type that we term tubular bone was also identified. This bone is completely filled with adipose tissue, unlike spongy bones. This study provides important insight on how osteogenesis takes place in zebrafish, and especially on the transition from cellular to acellular bones. Overall, this study leads to a deeper understanding of the functional histological composition of adult zebrafish bones.

  1. A conserved bacterial protein induces pancreatic beta cell expansion during zebrafish development

    PubMed Central

    Hill, Jennifer Hampton; Franzosa, Eric A; Huttenhower, Curtis; Guillemin, Karen

    2016-01-01

    Resident microbes play important roles in the development of the gastrointestinal tract, but their influence on other digestive organs is less well explored. Using the gnotobiotic zebrafish, we discovered that the normal expansion of the pancreatic β cell population during early larval development requires the intestinal microbiota and that specific bacterial members can restore normal β cell numbers. These bacteria share a gene that encodes a previously undescribed protein, named herein BefA (β Cell Expansion Factor A), which is sufficient to induce β cell proliferation in developing zebrafish larvae. Homologs of BefA are present in several human-associated bacterial species, and we show that they have conserved capacity to stimulate β cell proliferation in larval zebrafish. Our findings highlight a role for the microbiota in early pancreatic β cell development and suggest a possible basis for the association between low diversity childhood fecal microbiota and increased diabetes risk. DOI: http://dx.doi.org/10.7554/eLife.20145.001 PMID:27960075

  2. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

    PubMed Central

    Liu, Chao; Gates, Keith P.; Fang, Longhou; Amar, Marcelo J.; Schneider, Dina A.; Geng, Honglian; Huang, Wei; Kim, Jungsu; Pattison, Jennifer; Zhang, Jian; Witztum, Joseph L.; Remaley, Alan T.; Dong, P. Duc; Miller, Yury I.

    2015-01-01

    ABSTRACT Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases. PMID:26044956

  3. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia.

    PubMed

    Liu, Chao; Gates, Keith P; Fang, Longhou; Amar, Marcelo J; Schneider, Dina A; Geng, Honglian; Huang, Wei; Kim, Jungsu; Pattison, Jennifer; Zhang, Jian; Witztum, Joseph L; Remaley, Alan T; Dong, P Duc; Miller, Yury I

    2015-08-01

    Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases. © 2015. Published by The Company of Biologists Ltd.

  4. Craniofacial abnormalities result from knock down of nonsyndromic clefting gene, crispld2, in zebrafish

    PubMed Central

    Yuan, Qiuping; Chiquet, Brett T.; DeVault, Laura; Warman, Matthew L.; Nakamura, Yukio; Swindell, Eric C.; Hecht, Jacqueline T.

    2012-01-01

    Nonsyndromic cleft lip and palate (NSCLP), a common birth defect, affects 4000 newborns in the US each year. Previously, we described an association between CRISPLD2 and NSCLP and showed Crispld2 expression in the murine palate. These results suggested that a perturbation in CRISPLD2 activity affects craniofacial development. Here, we describe crispld2 expression and the phenotypic consequence of its loss of function in zebrafish. crispld2 was expressed at all stages of zebrafish morphogenesis examined and localized to the rostral end by 1-day post fertilization. Morpholino knockdown of crispld2 resulted in significant jaw and palatal abnormalities in a dose dependent manner. Loss of crispld2 caused aberrant patterning of neural crest cells (NCC) suggesting that crispld2 is necessary for normal NCC formation. Altogether, we show that crispld2 plays a significant role in the development of the zebrafish craniofacies and alteration of normal protein levels disturbs palate and jaw formation. These data provide support for a role of CRISPLD2 in NSCLP. PMID:22887593

  5. Hadp1, a newly identified pleckstrin homology domain protein, is required for cardiac contractility in zebrafish

    PubMed Central

    Wythe, Joshua D.; Jurynec, Michael J.; Urness, Lisa D.; Jones, Christopher A.; Sabeh, M. Khaled; Werdich, Andreas A.; Sato, Mariko; Yost, H. Joseph; Grunwald, David J.; MacRae, Calum A.; Li, Dean Y.

    2011-01-01

    SUMMARY The vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca2+ handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca2+ handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4)P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca2+ handling in the heart through its interaction with phosphatidylinositols. PMID:21628396

  6. Regeneration of Zebrafish CNS: Adult Neurogenesis

    PubMed Central

    Ghosh, Sukla; Hui, Subhra Prakash

    2016-01-01

    Regeneration in the animal kingdom is one of the most fascinating problems that have allowed scientists to address many issues of fundamental importance in basic biology. However, we came to know that the regenerative capability may vary across different species. Among vertebrates, fish and amphibians are capable of regenerating a variety of complex organs through epimorphosis. Zebrafish is an excellent animal model, which can repair several organs like damaged retina, severed spinal cord, injured brain and heart, and amputated fins. The focus of the present paper is on spinal cord regeneration in adult zebrafish. We intend to discuss our current understanding of the cellular and molecular mechanism(s) that allows formation of proliferating progenitors and controls neurogenesis, which involve changes in epigenetic and transcription programs. Unlike mammals, zebrafish retains radial glia, a nonneuronal cell type in their adult central nervous system. Injury induced proliferation involves radial glia which proliferate, transcribe embryonic genes, and can give rise to new neurons. Recent technological development of exquisite molecular tools in zebrafish, such as cell ablation, lineage analysis, and novel and substantial microarray, together with advancement in stem cell biology, allowed us to investigate how progenitor cells contribute to the generation of appropriate structures and various underlying mechanisms like reprogramming. PMID:27382491

  7. Teaching Stress Physiology Using Zebrafish ("Danio Rerio")

    ERIC Educational Resources Information Center

    Cooper, Michael; Dhawale, Shree; Mustafa, Ahmed

    2009-01-01

    A straightforward and inexpensive laboratory experiment is presented that investigates the physiological stress response of zebrafish after a 5 degree C increase in water temperature. This experiment is designed for an undergraduate physiology lab and allows students to learn the scientific method and relevant laboratory techniques without causing…

  8. Zebrafish in Toxicology and Environmental Health.

    PubMed

    Bambino, Kathryn; Chu, Jaime

    2017-01-01

    As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. © 2017 Elsevier Inc. All rights reserved.

  9. Zebrafish Embryo Model of Bartonella henselae Infection

    PubMed Central

    Lima, Amorce; Cha, Byeong J.; Amin, Jahanshah; Smith, Lisa K.

    2014-01-01

    Abstract Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)y1 zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis. PMID:25026365

  10. Zebrafish embryo model of Bartonella henselae infection.

    PubMed

    Lima, Amorce; Cha, Byeong J; Amin, Jahanshah; Smith, Lisa K; Anderson, Burt

    2014-10-01

    Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)(y1) zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.

  11. An outbreak of Plesimonus Shigelloides in Zebrafish

    USDA-ARS?s Scientific Manuscript database

    Plesiomonas shigelloides is a flagellated, gram-negative rod that is an emergent pathogen associated with human gastroenteritis. Recently, we experienced a disease outbreak in zebrafish that were obtained from a commercial source. Fourteen days after being held at 27°C in our flow-through quarantine...

  12. Nanomaterial Toxicity Screening in Developing Zebrafish Embryos

    EPA Science Inventory

    To assess nanomaterial vertebrate toxicity, a high-content screening assay was created using developing zebrafish, Danio rerio. This included a diverse group of nanomaterials (n=42 total) ranging from metallic (Ag, Au) and metal oxide (CeO2, CuO, TiO2, ZnO) nanoparticles, to non...

  13. Behavorial assessments of larval zebrafish neurotoxicology

    EPA Science Inventory

    Fishes have long been a popular organism in ecotoxicology research, and are increasingly used in human health research as an alternative animal model for chemical screening. Our laboratory incorporates a zebrafish (Danio rerio) embryo/larval assay to screen chemicals for developm...

  14. Zebrafish Locomotor Responses Predict Irritant Potential of ...

    EPA Pesticide Factsheets

    Over the past few decades, the drying and warming trends of global climate change have increased wildland fire (WF) season length, as well as geographic area impacted. Consequently, exposures to WF fine particulate matter (PM2.5; aerodynamic diameter <2.5 µm) are likely to increase in frequency and duration, contributing to a growing public health burden. Given the influence of fuel type and combustion conditions on WFPM2.5 composition, there is pressing need to identify the biomass fuel sources and emission constituents that drive toxicity. Previously, we reported the utility of 6-day post-fertilization (dpf) zebrafish larvae in evaluating diesel exhaust PM-induced irritation, demonstrating responses analogous to those in mammals. In the present study, combustions, separated by smoldering or flaming conditions, of pine needles, red oak, pine, eucalyptus, and peat were achieved using an automated tube furnace paired with a cryo-trapping apparatus to collect condensates of emissions. The condensates were extracted and prepared for use in zebrafish assays. We hypothesized that 1) the extractable organic fractions of biomass smoke PM will elicit dose-dependent irritant responses in 6-dpf zebrafish larvae, and 2) the relative potencies will vary across biomass emissions, potentially driven by varying chemical composition of fuel sources. Six-dpf zebrafish (n= 28-32/group) were exposed acutely to PM extracts (5 concentrations; 0.3-30 µg/ml; half-log intervals) and

  15. Detecting Developmental Neurotoxicants Using Zebrafish Embryos

    EPA Science Inventory

    As part of EPA’s program on the screening and prioritization of chemicals for developmental neurotoxicity, a rapid, cost-effective in vivo vertebrate screen is being developed using an alternative species approach. Zebrafish (Danio rerio), a small freshwater fish with external f...

  16. Somatic cell nuclear transfer in zebrafish.

    PubMed

    Siripattarapravat, Kannika; Pinmee, Boonya; Venta, Patrick J; Chang, Chia-Cheng; Cibelli, Jose B

    2009-10-01

    We developed a method for somatic cell nuclear transfer in zebrafish using laser-ablated metaphase II eggs as recipients, the micropyle for transfer of the nucleus and an egg activation protocol after nuclear reconstruction. We produced clones from cells of both embryonic and adult origins, although the latter did not give rise to live adult clones.

  17. Cadmium potentiates toxicity of cypermethrin in zebrafish.

    PubMed

    Yang, Ye; Ye, Xiaoqing; He, Buyuan; Liu, Jing

    2016-02-01

    Co-occurrence of pesticides such as synthetic pyrethroids and metals in aquatic ecosystems raises concerns over their combined ecological effects. Cypermethrin, 1 of the top 5 synthetic pyrethroids in use, has been extensively detected in surface water. Cadmium (Cd) has been recognized as 1 of the most toxic metals and is a common contaminant in the aquatic system. However, little information is available regarding their joint toxicity. In the present study, combined toxicity of cypermethrin and Cd and the underlying mechanisms were investigated. Zebrafish embryos and adults were exposed to the individual contaminant or binary mixtures. Co-exposure to cypermethrin and Cd produced synergistic effects on the occurrence of crooked body, pericardial edema, and noninflation of swim bladder. The addition of Cd significantly potentiated cypermethrin-induced spasms and caused more oxidative stress in zebrafish larvae. Cypermethrin-mediated induction of transcription levels and catalytic activities of cytochrome P450 (CYP) enzyme were significantly down-regulated by Cd in both zebrafish larvae and adults. Chemical analytical data showed that in vitro elimination of cypermethrin by CYP1A1 was inhibited by Cd. The addition of Cd caused an elevation of in vivo cypermethrin residue levels in the mixture-exposed adult zebrafish. These results suggest that the enhanced toxicity of cypermethrin in the presence of Cd results from the inhibitory effects of Cd on CYP-mediated biotransformation of this pesticide. The authors' findings provide a deeper understanding of the mechanistic basis accounting for the joint toxicity of cypermethrin and Cd.

  18. Behavorial assessments of larval zebrafish neurotoxicology

    EPA Science Inventory

    Fishes have long been a popular organism in ecotoxicology research, and are increasingly used in human health research as an alternative animal model for chemical screening. Our laboratory incorporates a zebrafish (Danio rerio) embryo/larval assay to screen chemicals for developm...

  19. Nanomaterial Toxicity Screening in Developing Zebrafish Embryos

    EPA Science Inventory

    To assess nanomaterial vertebrate toxicity, a high-content screening assay was created using developing zebrafish, Danio rerio. This included a diverse group of nanomaterials (n=42 total) ranging from metallic (Ag, Au) and metal oxide (CeO2, CuO, TiO2, ZnO) nanoparticles, to non...

  20. Bioenergetic Profiling of Zebrafish Embryonic Development

    PubMed Central

    Stackley, Krista D.; Beeson, Craig C.; Rahn, Jennifer J.; Chan, Sherine S. L.

    2011-01-01

    Many debilitating conditions are linked to bioenergetic defects. Developing screens to probe the genetic and/or chemical basis for such links has proved intractable. Furthermore, there is a need for a physiologically relevant assay of bioenergetics in whole organisms, especially for early stages in life where perturbations could increase disease susceptibility with aging. Thus, we asked whether we could screen bioenergetics and mitochondrial function in the developing zebrafish embryo. We present a multiplexed method to assay bioenergetics in zebrafish embryos from the blastula period (3 hours post-fertilization, hpf) through to hatching (48 hpf). In proof of principle experiments, we measured respiration and acid extrusion of developing zebrafish embryos. We quantified respiratory coupling to various bioenergetic functions by using specific pharmacological inhibitors of bioenergetic pathways. We demonstrate that changes in the coupling to ATP turnover and proton leak are correlated with developmental stage. The multiwell format of this assay enables the user to screen for the effects of drugs and environmental agents on bioenergetics in the zebrafish embryo with high sensitivity and reproducibility. PMID:21980518

  1. Zebrafish phenotypic screen identifies novel Notch antagonists.

    PubMed

    Velaithan, Vithya; Okuda, Kazuhide Shaun; Ng, Mei Fong; Samat, Norazwana; Leong, Sze Wei; Faudzi, Siti Munirah Mohd; Abas, Faridah; Shaari, Khozirah; Cheong, Sok Ching; Tan, Pei Jean; Patel, Vyomesh

    2017-04-01

    Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27(KIP1). Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

  2. Nicotine response genetics in the zebrafish

    PubMed Central

    Petzold, Andrew M.; Balciunas, Darius; Sivasubbu, Sridhar; Clark, Karl J.; Bedell, Victoria M.; Westcot, Stephanie E.; Myers, Shelly R.; Moulder, Gary L.; Thomas, Mark J.; Ekker, Stephen C.

    2009-01-01

    Tobacco use is predicted to result in over 1 billion deaths worldwide by the end of the 21st century. How genetic variation contributes to the observed differential predisposition in the human population to drug dependence is unknown. The zebrafish (Danio rerio) is an emerging vertebrate model system for understanding the genetics of behavior. We developed a nicotine behavioral assay in zebrafish and applied it in a forward genetic screen using gene-breaking transposon mutagenesis. We used this method to molecularly characterize bdav/cct8 and hbog/gabbr1.2 as mutations with altered nicotine response. Each have a single human ortholog, identifying two points for potential scientific, diagnostic, and drug development for nicotine biology and cessation therapeutics. We show this insertional method generates mutant alleles that are reversible through Cre-mediated recombination, representing a conditional mutation system for the zebrafish. The combination of this reporter-tagged insertional mutagen approach and zebrafish provides a powerful platform for a rich array of questions amenable to genetic-based scientific inquiry, including the basis of behavior, epigenetics, plasticity, stress, memory, and learning. PMID:19858493

  3. Detecting Developmental Neurotoxicants Using Zebrafish Embryos

    EPA Science Inventory

    As part of EPA’s program on the screening and prioritization of chemicals for developmental neurotoxicity, a rapid, cost-effective in vivo vertebrate screen is being developed using an alternative species approach. Zebrafish (Danio rerio), a small freshwater fish with external f...

  4. Molecular cloning and developmental expression of foxP2 in zebrafish.

    PubMed

    Bonkowsky, Joshua L; Chien, Chi-Bin

    2005-11-01

    Forkhead domain transcription factors are a large gene family with multiple roles in development. FOXP2, a recently identified member of this family, has been shown to be critical for normal development of language in humans, but little is known of its broader function during nervous system development. We report here the cloning of foxP2, the zebrafish ortholog of FOXP2. Zebrafish FoxP2 is highly conserved in its zinc-finger and forkhead domains, but lacks the large glutamine repeat characteristic of its orthologs. In examining the spatial and temporal distribution of foxP2 during development, we find that it is specifically expressed in many domains of the nervous system, including the telencephalon, diencephalon, cerebellum, hindbrain, tectum, retinal ganglion cells, and spinal cord. Thus, in addition to specific roles in language development, foxP2 likely has a more general conserved role in nervous system development.

  5. A non-canonical function of telomerase RNA in the regulation of developmental myelopoiesis in zebrafish

    NASA Astrophysics Data System (ADS)

    Alcaraz-Pérez, Francisca; García-Castillo, Jesús; García-Moreno, Diana; López-Muñoz, Azucena; Anchelin, Monique; Angosto, Diego; Zon, Leonard I.; Mulero, Victoriano; Cayuela, María L.

    2014-02-01

    Dyskeratosis congenita (DC) is an inherited disorder with mutations affecting telomerase or telomeric proteins. DC patients usually die of bone marrow failure. Here we show that genetic depletion of the telomerase RNA component (TR) in the zebrafish results in impaired myelopoiesis, despite normal development of haematopoietic stem cells (HSCs). The neutropenia caused by TR depletion is independent of telomere length and telomerase activity. Genetic analysis shows that TR modulates the myeloid-erythroid fate decision by controlling the levels of the master myeloid and erythroid transcription factors spi1 and gata1, respectively. The alteration in spi1 and gata1 levels occurs through stimulation of gcsf and mcsf. Our model of TR deficiency in the zebrafish illuminates the non-canonical roles of TR, and could establish therapeutic targets for DC.

  6. Identification of Fusarium solani species complex from infected zebrafish (Danio rerio).

    PubMed

    Ke, Xiaoli; Lu, Maixin; Wang, Jianguo

    2016-11-01

    Although Fusarium sp. infections have been reported in aquatic invertebrates, studies of Fusarium spp. as fish pathogens remain very limited. In our study, a fungus was isolated from diseased zebrafish (Danio rerio). DNA sequence analysis of the fungus, based on a partial region of the translation elongation factor 1α gene (EF-1α), the internal transcribed spacer region and domains D1 and D2 of the large subunit of the ribosomal RNA gene (ITS plus LSU), and the RNA polymerase II subunit gene (RPB2), showed 99.9-100% homology to Fusarium solani species complex sequences. Multilocus sequence typing analysis based on 3-locus haplotypes (EF-1α, ITS plus LSU, and RPB2) suggests that the isolated strain was type 3+4-P. Challenge experiments showed that this organism could be pathogenic to zebrafish, but usually does not infect healthy subjects under normal circumstances.

  7. Histone deacetylase 1 is required for the development of the zebrafish inner ear

    PubMed Central

    He, Yingzi; Tang, Dongmei; Li, Wenyan; Chai, Renjie; Li, Huawei

    2016-01-01

    Histone deacetylase 1 (HDAC1) has been reported to be important for multiple aspects of normal embryonic development, but little is known about its function in the development of mechanosensory organs. Here, we first confirmed that HDAC1 is expressed in the developing otic vesicles of zebrafish by whole-mount in situ hybridization. Knockdown of HDAC1 using antisense morpholino oligonucleotides in zebrafish embryos induced smaller otic vesicles, abnormal otoliths, malformed or absent semicircular canals, and fewer sensory hair cells. HDAC1 loss of function also caused attenuated expression of a subset of key genes required for otic vesicle formation during development. Morpholino-mediated knockdown of HDAC1 resulted in decreased expression of members of the Fgf family in the otic vesicles, suggesting that HDAC1 is involved in the development of the inner ear through regulation of Fgf signaling pathways. Taken together, our results indicate that HDAC1 plays an important role in otic vesicle formation. PMID:26832938

  8. Involvement of the α1-adrenoceptor in sleep-waking and sleep loss-induced anxiety behavior in zebrafish.

    PubMed

    Singh, A; Subhashini, N; Sharma, S; Mallick, B N

    2013-08-15

    Sleep is a universal phenomenon in vertebrates, and its loss affects various behaviors. Independent studies have reported that sleep loss increases anxiety; however, the detailed mechanism is unknown. Because sleep deprivation increases noradrenalin (NA), which modulates many behaviors and induces patho-physiological changes, this study utilized zebrafish as a model to investigate whether sleep loss-induced increased anxiety is modulated by NA. Continuous behavioral quiescence for at least 6s was considered to represent sleep in zebrafish; although some authors termed it as a sleep-like state, in this study we have termed it as sleep. The activity of fish that signified sleep-waking was recorded in light-dark, during continuous dark and light; the latter induced sleep loss in fish. The latency, number of entries, time spent and distance travelled in the light chamber were assessed in a light-dark box test to estimate the anxiety behavior of normal, sleep-deprived and prazosin (PRZ)-treated fish. Zebrafish showed increased waking during light and complete loss of sleep upon continuous exposure to light for 24h. PRZ significantly increased sleep in normal fish. Sleep-deprived fish showed an increased preference for dark (expression of increased anxiety), and this effect was prevented by PRZ, which increased sleep as well. Our findings suggest that sleep loss-induced anxiety-like behavior in zebrafish is likely to be mediated by NA's action on the α1-adrenoceptor.

  9. Intravital imaging of metastasis in adult Zebrafish.

    PubMed

    Benjamin, David C; Hynes, Richard O

    2017-09-25

    Metastasis is a major clinical problem whose biology is not yet fully understood. This lack of understanding is especially true for the events at the metastatic site, which include arrest, extravasation, and growth into macrometastases. Intravital imaging is a powerful technique that has shown great promise in increasing our understanding of these events. To date, most intravital imaging studies have been performed in mice, which has limited its adoption. Zebrafish are also a common system for the intravital imaging of metastasis. However, as imaging in embryos is technically simpler, relatively few studies have used adult zebrafish to study metastasis and none have followed individual cells at the metastatic site over time. The aim of this study was to demonstrate that adult casper zebrafish offer a convenient model system for performing intravital imaging of the metastatic site over time with single-cell resolution. ZMEL1 zebrafish melanoma cells were injected into 6 to 10-week-old casper fish using an intravenous injection protocol. Because casper fish are transparent even as adults, they could be imaged without surgical intervention. Individual cells were followed over the course of 2 weeks as they arrested, extravasated, and formed macroscopic metastases. Our injection method reliably delivered cells into circulation and led to the formation of tumors in multiple organs. Cells in the skin and sub-dermal muscle could be imaged at high resolution over 2 weeks using confocal microscopy. Arrest was visualized and determined to be primarily due to size restriction. Following arrest, extravasation was seen to occur between 1 and 6 days post-injection. Once outside of the vasculature, cells were observed migrating as well as forming protrusions. Casper fish are a useful model for studying the events at the metastatic site using intravital imaging. The protocols described in this study are relatively simple. Combined with the reasonably low cost of zebrafish, they

  10. Defects of the Glycinergic Synapse in Zebrafish

    PubMed Central

    Ogino, Kazutoyo; Hirata, Hiromi

    2016-01-01

    Glycine mediates fast inhibitory synaptic transmission. Physiological importance of the glycinergic synapse is well established in the brainstem and the spinal cord. In humans, the loss of glycinergic function in the spinal cord and brainstem leads to hyperekplexia, which is characterized by an excess startle reflex to sudden acoustic or tactile stimulation. In addition, glycinergic synapses in this region are also involved in the regulation of respiration and locomotion, and in the nociceptive processing. The importance of the glycinergic synapse is conserved across vertebrate species. A teleost fish, the zebrafish, offers several advantages as a vertebrate model for research of glycinergic synapse. Mutagenesis screens in zebrafish have isolated two motor defective mutants that have pathogenic mutations in glycinergic synaptic transmission: bandoneon (beo) and shocked (sho). Beo mutants have a loss-of-function mutation of glycine receptor (GlyR) β-subunit b, alternatively, sho mutant is a glycinergic transporter 1 (GlyT1) defective mutant. These mutants are useful animal models for understanding of glycinergic synaptic transmission and for identification of novel therapeutic agents for human diseases arising from defect in glycinergic transmission, such as hyperekplexia or glycine encephalopathy. Recent advances in techniques for genome editing and for imaging and manipulating of a molecule or a physiological process make zebrafish more attractive model. In this review, we describe the glycinergic defective zebrafish mutants and the technical advances in both forward and reverse genetic approaches as well as in vivo visualization and manipulation approaches for the study of the glycinergic synapse in zebrafish. PMID:27445686

  11. Zic1 and Zic4 regulate zebrafish roof plate specification and hindbrain ventricle morphogenesis

    PubMed Central

    Elsen, Gina E.; Choi, Louis; Millen, Kathleen; Grinblat, Yevgenya; Prince, Victoria E.

    2008-01-01

    During development, the lumen of the neural tube develops into a system of brain cavities or ventricles, which play important roles in normal CNS function. We have established that the formation of the hindbrain (4th) ventricle in zebrafish is dependent upon the pleiotropic functions of the genes implicated in human Dandy Walker Malformation, Zic1 and Zic4. Using morpholino knockdown we show that zebrafish Zic1 and Zic4 are required for normal morphogenesis of the 4th ventricle. In Zic1 and/or Zic4 morphants the ventricle does not open properly, but remains completely or partially fused from the level of rhombomere (r) 2 towards the posterior. In the absence of Zic function early hindbrain regionalization and neural crest development remain unaffected, but dorsal hindbrain progenitor cell proliferation is significantly reduced. Importantly, we find that Zic1 and Zic4 are required for development of the dorsal roof plate. In Zic morphants expression of roof plate markers, including lmx1b.1 and lmx1b.2, is disrupted. We further demonstrate that zebrafish Lmx1b function is required for both hindbrain roof plate development and 4th ventricle morphogenesis, confirming that roof plate formation is a critical component of ventricle development. Finally, we show that dorsal rhombomere boundary signaling centers depend on Zic1 and Zic4 function and on roof plate signals, and provide evidence that these boundary signals are also required for ventricle morphogenesis. In summary, we conclude that Zic1 and Zic4 control zebrafish 4th ventricle morphogenesis by regulating multiple mechanisms including cell proliferation and fate specification in the dorsal hindbrain. PMID:18191121

  12. Rapid, accurate, and non-invasive measurement of zebrafish axial length and other eye dimensions using SD-OCT allows longitudinal analysis of myopia and emmetropization.

    PubMed

    Collery, Ross F; Veth, Kerry N; Dubis, Adam M; Carroll, Joseph; Link, Brian A

    2014-01-01

    Refractive errors in vision can be caused by aberrant axial length of the eye, irregular corneal shape, or lens abnormalities. Causes of eye length overgrowth include multiple genetic loci, and visual parameters. We evaluate zebrafish as a potential animal model for studies of the genetic, cellular, and signaling basis of emmetropization and myopia. Axial length and other eye dimensions of zebrafish were measured using spectral domain-optical coherence tomography (SD-OCT). We used ocular lens and body metrics to normalize and compare eye size and relative refractive error (difference between observed retinal radial length and controls) in wild-type and lrp2 zebrafish. Zebrafish were dark-reared to assess effects of visual deprivation on eye size. Two relative measurements, ocular axial length to body length and axial length to lens diameter, were found to accurately normalize comparisons of eye sizes between different sized fish (R2=0.9548, R2=0.9921). Ray-traced focal lengths of wild-type zebrafish lenses were equal to their retinal radii, while lrp2 eyes had longer retinal radii than focal lengths. Both genetic mutation (lrp2) and environmental manipulation (dark-rearing) caused elongated eye axes. lrp2 mutants had relative refractive errors of -0.327 compared to wild-types, and dark-reared wild-type fish had relative refractive errors of -0.132 compared to light-reared siblings. Therefore, zebrafish eye anatomy (axial length, lens radius, retinal radius) can be rapidly and accurately measured by SD-OCT, facilitating longitudinal studies of regulated eye growth and emmetropization. Specifically, genes homologous to human myopia candidates may be modified, inactivated or overexpressed in zebrafish, and myopia-sensitizing conditions used to probe gene-environment interactions. Our studies provide foundation for such investigations into genetic contributions that control eye size and impact refractive errors.

  13. Behavioral analysis of zebrafish larvae swimming in three dimensions

    NASA Astrophysics Data System (ADS)

    Feng, Ruopei; Girdhar, Kiran; Chemla, Yann; Gruebele, Martin

    2015-03-01

    Behavioral biologists have a strong interest in studying the behavior of larval zebrafish because the limited number of locomotor neurons in larval zebrafish couples with the rich repertoire of movements as a vertebrate animal. Current research uses a priori-selected parameters to describe their movements. Most research also only considers the 2D movements of zebrafish, leaving out the vertical component of their locomotion. Our lab has developed a method to reduce the dimensionality of the locomotion of zebrafish and determine the behavioral space of 2D swimming. We are extending this work to capture 3D locomotion of zebrafish larvae. Here we present our preliminary analysis of the 3D locomotion of zebrafish.

  14. Conserved gene regulation during acute inflammation between zebrafish and mammals.

    PubMed

    Forn-Cuní, G; Varela, M; Pereiro, P; Novoa, B; Figueras, A

    2017-02-03

    Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential.

  15. Zebrafish Xenograft: An Evolutionary Experiment in Tumour Biology.

    PubMed

    Wyatt, Rachael A; Trieu, Nhu P V; Crawford, Bryan D

    2017-09-05

    Though the cancer research community has used mouse xenografts for decades more than zebrafish xenografts, zebrafish have much to offer: they are cheap, easy to work with, and the embryonic model is relatively easy to use in high-throughput assays. Zebrafish can be imaged live, allowing us to observe cellular and molecular processes in vivo in real time. Opponents dismiss the zebrafish model due to the evolutionary distance between zebrafish and humans, as compared to mice, but proponents argue for the zebrafish xenograft's superiority to cell culture systems and its advantages in imaging. This review places the zebrafish xenograft in the context of current views on cancer and gives an overview of how several aspects of this evolutionary disease can be addressed in the zebrafish model. Zebrafish are missing homologs of some human proteins and (of particular interest) several members of the matrix metalloproteinase (MMP) family of proteases, which are known for their importance in tumour biology. This review draws attention to the implicit evolutionary experiment taking place when the molecular ecology of the xenograft host is significantly different than that of the donor.

  16. Deriving cell lines from zebrafish embryos and tumors.

    PubMed

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  17. Biologically inspired robots elicit a robust fear response in zebrafish

    NASA Astrophysics Data System (ADS)

    Ladu, Fabrizio; Bartolini, Tiziana; Panitz, Sarah G.; Butail, Sachit; Macrı, Simone; Porfiri, Maurizio

    2015-03-01

    We investigate the behavioral response of zebrafish to three fear-evoking stimuli. In a binary choice test, zebrafish are exposed to a live allopatric predator, a biologically-inspired robot, and a computer-animated image of the live predator. A target tracking algorithm is developed to score zebrafish behavior. Unlike computer-animated images, the robotic and live predator elicit a robust avoidance response. Importantly, the robotic stimulus elicits more consistent inter-individual responses than the live predator. Results from this effort are expected to aid in hypothesis-driven studies on zebrafish fear response, by offering a valuable approach to maximize data-throughput and minimize animal subjects.

  18. The Cardiac Transcriptome and Dilated Cardiomyopathy Genes in Zebrafish

    PubMed Central

    Shih, Yu-Huan; Zhang, Yuji; Ding, Yonghe; Ross, Christian A.; Li, Hu; Olson, Timothy M.; Xu, Xiaolei

    2015-01-01

    Background Genetic studies of cardiomyopathy and heart failure have limited throughput in mammalian models. Adult zebrafish have been recently pursued as a vertebrate model with higher throughput, but genetic conservation must be tested. Methods and Results We conducted transcriptome analysis of zebrafish heart and searched for fish homologues of 51 known human dilated cardiomyopathy (DCM)-associated genes. We also identified genes with high cardiac expression and genes with differential expression between embryonic and adult stages. Among tested genes, 30 had a single zebrafish orthologue, 14 had 2 homologues, and 5 had 3 or more homologues. By analyzing the expression data on the basis of cardiac abundance and enrichment hypotheses, we identified a single zebrafish gene for 14 of 19 multiple-homologue genes and 2 zebrafish homologues of high priority for ACTC1. Of note, our data suggested vmhc and vmhcl as functional zebrafish orthologues for human MYH6 and MYH7, respectively, which are established molecular markers for cardiac remodeling. Conclusions Most known genes for human DCM have a corresponding zebrafish orthologue, which supports the use of zebrafish as a conserved vertebrate model. Definition of the cardiac transcriptome and fetal gene program will facilitate systems biology studies of DCM in zebrafish. PMID:25583992

  19. The Zebrafish Anatomy Portal: a novel integrated resource to facilitate zebrafish research.

    PubMed

    Salgado, David; Marcelle, Christophe; Currie, Peter D; Bryson-Richardson, Robert J

    2012-12-01

    Zebrafish is a common model organism in research and yet, despite its widespread use, anatomical resources for this species are incomplete or lacking in functionality. There remains a need for a single reference resource that integrates user-friendly tools to facilitate the identification of structures, display of reference images, provides data on gene expression, links to relevant literature, and covers the complete range of zebrafish developmental stages. To fulfill this need, we have designed the Zebrafish Anatomy Portal (www.zfap.org), containing annotated three-dimensional images of zebrafish at stages throughout development and adulthood, acquired by optical projection tomography. ZFAP combines functionalities to allow scanning through 3D data sets, searching of images by anatomical terms, predictions of gene expression from literature analysis, and facilitation of the identification of relevant literature through assisted searching of the NCBI PubMed resource. ZFAP provides a highly functional anatomical resource that will aid future education and research in the zebrafish model system. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Bigh3 is upregulated in regenerating zebrafish fin.

    PubMed

    Page, Lionel; Polok, Bozena; Bustamante, Mauro; Schorderet, Daniel F

    2013-03-01

    Zebrafish is a good model for studying regeneration because of the rapidity with which it occurs. Better understanding of this process may lead in the future to improvement of the regenerating capacity of humans. Signaling factors are the second largest category of genes, regulated during regeneration after the regulators of wound healing. Major developmental signaling pathways play a role in this multistep process, such as Bmp, Fgf, Notch, retinoic acid, Shh, and Wnt. In the present study, we focus on TGF-β-induced genes, bigh3 and bambia. Bigh3 encodes keratoepithelin, a protein first identified as an extracellular matrix protein reported to play a role in cell adhesion, as well as in cornea formation and osteogenesis. The expression of bigh3 in zebrafish fins has previously been reported. Here we demonstrate that tgf-b1 and tgf-b3 mRNA reacted with delay, first showing no regulation at 3 dpa, followed by upregulation at 4 and 5 dpa. Tgf-b1, tgf-2, and tgf-brII mRNA were back to normal levels at 10 dpa. Only tgf-b3 mRNA was still upregulated at that time. Bigh3 mRNA followed the upregulation of tgf-b1, while bambia mRNA behaved similarly to tgf-b2 mRNA. We show that upregulation of bigh3 and bambia mRNA correlated with the process of fin regeneration and regulation of TGF-b signaling, suggesting a new role for these proteins.

  1. Progenitor potential of nkx6.1-expressing cells throughout zebrafish life and during beta cell regeneration.

    PubMed

    Ghaye, Aurélie P; Bergemann, David; Tarifeño-Saldivia, Estefania; Flasse, Lydie C; Von Berg, Virginie; Peers, Bernard; Voz, Marianne L; Manfroid, Isabelle

    2015-09-02

    In contrast to mammals, the zebrafish has the remarkable capacity to regenerate its pancreatic beta cells very efficiently. Understanding the mechanisms of regeneration in the zebrafish and the differences with mammals will be fundamental to discovering molecules able to stimulate the regeneration process in mammals. To identify the pancreatic cells able to give rise to new beta cells in the zebrafish, we generated new transgenic lines allowing the tracing of multipotent pancreatic progenitors and endocrine precursors. Using novel bacterial artificial chromosome transgenic nkx6.1 and ascl1b reporter lines, we established that nkx6.1-positive cells give rise to all the pancreatic cell types and ascl1b-positive cells give rise to all the endocrine cell types in the zebrafish embryo. These two genes are initially co-expressed in the pancreatic primordium and their domains segregate, not as a result of mutual repression, but through the opposite effects of Notch signaling, maintaining nkx6.1 expression while repressing ascl1b in progenitors. In the adult zebrafish, nkx6.1 expression persists exclusively in the ductal tree at the tip of which its expression coincides with Notch active signaling in centroacinar/terminal end duct cells. Tracing these cells reveals that they are able to differentiate into other ductal cells and into insulin-expressing cells in normal (non-diabetic) animals. This capacity of ductal cells to generate endocrine cells is supported by the detection of ascl1b in the nkx6.1:GFP ductal cell transcriptome. This transcriptome also reveals, besides actors of the Notch and Wnt pathways, several novel markers such as id2a. Finally, we show that beta cell ablation in the adult zebrafish triggers proliferation of ductal cells and their differentiation into insulin-expressing cells. We have shown that, in the zebrafish embryo, nkx6.1+ cells are bona fide multipotent pancreatic progenitors, while ascl1b+ cells represent committed endocrine precursors. In

  2. Silver Nanoparticle Toxicity in the Embryonic Zebrafish is Governed by Particle Dispersion and Ionic Environment

    PubMed Central

    Wehmas, Leah; Tanguay, Robert L.

    2013-01-01

    The mechanism of action of silver nanoparticles (AgNPs) is unclear due to the particles’ strong tendency to agglomerate. Preventing agglomeration could offer precise control of the physicochemical properties that drive biological response to AgNPs. In an attempt to control agglomeration, we exposed zebrafish embryos to AgNPs of 20 or 110 nm core size, and polypyrrolidone (PVP) or citrate surface coatings in media of varying ionic strength. AgNPs remained unagglomerated in 62.5 μM CaCl2 (CaCl2) and ultrapure water (UP), but not in standard zebrafish embryo medium (EM). Zebrafish embryos developed normally in the low ionic strength environments of CaCl2 and UP. Exposure of embryos to AgNPs suspended in UP and CaCl2 resulted in higher toxicity than suspensions in EM. 20 nm AgNPs were more toxic than 110 nm AgNPs, and the PVP coating was more toxic than the citrate coating at the same particle core size. The silver tissue burden correlated well with observed toxicity but only for those exposures where the AgNPs remained unagglomerated. Our results demonstrate that size- and surface coating-dependent toxicity is a result of AgNPs remaining unagglomerated, and thus a critical-design consideration for experiments to offer meaningful evaluations of AgNP toxicity. PMID:23449170

  3. Imaging Beta Cell Regeneration and Interactions with Islet Vasculature in Transparent Adult Zebrafish

    PubMed Central

    Moss, Larry G.; Caplan, Tanner V.

    2013-01-01

    Abstract Blood vessel networks provide nutrients and gaseous exchange that are essential for functions. Pancreatic islet capillaries deliver oxygen to endocrine cells while transporting hormones to organs and peripheral locations throughout the body. We have developed a zebrafish diabetes model in which adult islets can be followed in vivo during beta cell regeneration while calibrating changes in beta cell mass and fasting blood glucose levels. After genetic ablation, beta cells are initially dysfunctional or dying, and blood glucose levels increase fourfold. During a 2-week period, hyperglycemia eventually normalizes as beta cell mass regenerates. We show that mCherry-fluorescent, insulin-positive beta cells re-emerge in close contact with the vascular endothelium. Alterations in the dense vascular network of zebrafish islets were visualized by the expression of green fluorescent protein (GFP) in endothelial cells derived from the Fli transcription factor promoter. The rapid destruction and regeneration of beta cell mass was evaluated in the same animal over time, providing a functional model for investigating the interactions of islet cell types with vascular cells as well as the consequences of hyperglycemia on other tissues. Regenerating adult zebrafish can be utilized as vertebrate, metabolically active models for generating new insights into treatments for type 2 diabetes. PMID:23682836

  4. Amigo adhesion protein regulates development of neural circuits in zebrafish brain.

    PubMed

    Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

    2014-07-18

    The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Silver nanoparticle toxicity in the embryonic zebrafish is governed by particle dispersion and ionic environment

    NASA Astrophysics Data System (ADS)

    Kim, Ki-Tae; Truong, Lisa; Wehmas, Leah; Tanguay, Robert L.

    2013-03-01

    The mechanism of action of silver nanoparticles (AgNPs) is unclear due to the particles’ strong tendency to agglomerate. Preventing agglomeration could offer precise control of the physicochemical properties that drive biological response to AgNPs. In an attempt to control agglomeration, we exposed zebrafish embryos to AgNPs of 20 or 110 nm core size, and polypyrrolidone (PVP) or citrate surface coatings in media of varying ionic strength. AgNPs remained unagglomerated in 62.5 μM CaCl2 (CaCl2) and ultrapure water (UP), but not in standard zebrafish embryo medium (EM). Zebrafish embryos developed normally in the low ionic strength environments of CaCl2 and UP. Exposure of embryos to AgNPs suspended in UP and CaCl2 resulted in higher toxicity than suspensions in EM. 20 nm AgNPs were more toxic than 110 nm AgNPs, and the PVP coating was more toxic than the citrate coating at the same particle core size. The silver tissue burden correlated well with observed toxicity but only for those exposures where the AgNPs remained unagglomerated. Our results demonstrate that size- and surface coating-dependent toxicity is a result of AgNPs remaining unagglomerated, and thus a critical-design consideration for experiments to offer meaningful evaluations of AgNP toxicity.

  6. The microcephaly gene aspm is involved in brain development in zebrafish

    SciTech Connect

    Kim, Hyun-Taek; Lee, Mi-Sun; Choi, Jung-Hwa; Jung, Ju-Yeon; Ahn, Dae-Gwon; Yeo, Sang-Yeob; Choi, Dong-Kug; Kim, Cheol-Hee

    2011-06-17

    Highlights: {yields} We identified a zebrafish aspm/mcph5 gene that is expressed in proliferating cells in the CNS during early development. {yields} Embryos injected with the aspm MO consistently showed a reduced head and eye size but were otherwise grossly normal, closely mimicking the known phenotypes of human microcephaly patients. {yields} Knock-down of aspm causes cell cycle arrest and apoptotic cell death during early development. -- Abstract: MCPH is a neurodevelopmental disorder characterized by a global reduction in cerebral cortical volume. Homozygous mutation of the MCPH5 gene, also known as ASPM, is the most common cause of the MCPH phenotype. To elucidate the roles of ASPM during embryonic development, the zebrafish aspm was identified, which is specifically expressed in proliferating cells in the CNS. Morpholino-mediated knock-down of aspm resulted in a significant reduction in head size. Furthermore, aspm-deficient embryos exhibited a mitotic arrest during early development. These findings suggest that the reduction in brain size in MCPH might be caused by lack of aspm function in the mitotic cell cycle and demonstrate that the zebrafish can provide a model system for congenital diseases of the human nervous system.

  7. Growth and maturation in the zebrafish, Danio rerio: a staging tool for teaching and research.

    PubMed

    Singleman, Corinna; Holtzman, Nathalia G

    2014-08-01

    Zebrafish have been increasingly used as a teaching tool to enhance the learning of many biological concepts from genetics, development, and behavior to the understanding of the local watershed. Traditionally, in both research and teaching, zebrafish work has focused on embryonic stages; however, later stages, from larval through adulthood, are increasingly being examined. Defining developmental stages based on age is a problematic way to assess maturity, because many environmental factors, such as temperature, population density, and water quality, impact growth and maturation. Fish length and characterization of key external morphological traits are considered better markers for maturation state. While a number of staging series exist for zebrafish, here we present a simplified normalization table of post-embryonic maturation well suited to both educational and research use. Specifically, we utilize fish size and four easily identified external morphological traits (pigment pattern, tail fin, anal fin, and dorsal fin morphology) to describe three larval stages, a juvenile stage, and an adult stage. These simplified maturation standards will be a useful tool for both educational and research protocols.

  8. Clonal origins of cells in the pigmented retina of the zebrafish eye

    SciTech Connect

    Streisinger, G.; Coale, F.; Taggart, C.; Walker, C.; Grunwald, D.J.

    1989-01-01

    Mosaic analysis has been used to study the clonal basis of the development of the pigmented retina of the zebrafish, Brachydanio rerio. Zebrafish embryos heterozygous for a recessive mutation at the gol-1 locus were exposed to gamma-irradiation at various developmental stages to create mosaic individuals consisting of wild-type pigmented cells and a clone of pigmentless (golden) cells in the eye. The contribution of individual embryonic cells to the pigmented retina was measured and the total number of cells in the embryo that contributed descendants to this tissue was determined. Until the 32-cell stage, almost every blastomere has some descendants that participate in the formation of the pigmented retina of the zebrafish. During subsequent cell divisions, up to the several thousand-cell stage, the number of ancestral cells is constant: approximately 40 cells are present that will give rise to progeny in the pigmented retina. Analysis of the size of clones in the pigmented retina indicates that the cells of this tissue do not arise through a rigid series of cell divisions originating in the early embryo. The findings that each cleavage stage cell contributes to the pigmented retina and yet the contribution of such cells is highly variable are consistent with the interpretation that clonal descendants of different blastomeres normally intermix extensively prior to formation of the pigmented retina.

  9. Behavioral and synaptic circuit features in a zebrafish model of fragile X syndrome.

    PubMed

    Ng, Ming-Chong; Yang, Yi-Ling; Lu, Kwok-Tung

    2013-01-01

    Fragile X syndrome (FXS) is the most frequent inherited form of human mental retardation. It is characterized by cognitive impairment and physical and behavioral problems and is caused by the silencing of fmr1 transcription and the absence of the fmr1 protein (FMRP). Recently, animal models of FXS have greatly facilitated the investigation of the molecular and cellular mechanisms of this loss-of-function disorder. The present study was aimed to further characterize the role of FMRP in behavior and synaptic function by using fmr1 knockout zebrafish. In adult zebrafish, we found that fmr1 knockout produces the anxiolytic-like responses of increased exploratory behavior in light/dark and open-field tests and avoidance learning impairment. Furthermore, electrophysiological recordings from telencephalic slice preparations of knockout fish displayed markedly reduced long-term potentiation and enhanced long-term depression compared to wild-type fish; however, basal glutamatergic transmission and presynaptic function at the lateral (Dl) and medial (Dm) division of the dorsal telencephalon synapse remained normal. Taken together, our study not only evaluates the mechanism of FRMP but also suggests that zebrafish have valuable potential as a complementary vertebrate model in studying the molecular pathogenesis of human fragile X syndrome.

  10. Identification and developmental expression pattern of van gogh-like 1, a second zebrafish strabismus homologue.

    PubMed

    Jessen, Jason R; Solnica-Krezel, Lilianna

    2004-05-01

    Cell movement plays a central role in both normal embryogenesis and the development of diseases such as cancer. Therefore, identification and analysis of proteins controlling cell movement is of special importance. The zebrafish trilobite locus encodes a Van Gogh/Strabismus homologue, which regulates diverse cell migratory behaviors during embryogenesis. Trilobite is most similar to human Van Gogh-like 2 (VANGL2)/Strabismus 1 and mouse Loop-tail associated protein/Lpp1. Both human and mouse genomes encode a second Strabismus homologue referred to as VANGL1/Strabismus 2 and Lpp2, respectively. This prompted us to ask whether another van gogh/strabismus gene, one more closely related to human VANGL1, exists in the zebrafish genome. This paper describes the identification of zebrafish vangl1 and provides the first spatiotemporal expression and functional analysis of a vertebrate vangl1 homologue. Our data indicate that vangl1 and trilobite/vangl2 are expressed in largely non-overlapping domains during embryogenesis. Injection of synthetic vangl1 RNA partially suppressed the gastrulation defect in trilobite mutant embryos, suggesting that Vangl1 and Trilobite/Vangl2 have similar biochemical activities.

  11. Growth and Maturation in the Zebrafish, Danio Rerio: A Staging Tool for Teaching and Research

    PubMed Central

    Singleman, Corinna

    2014-01-01

    Abstract Zebrafish have been increasingly used as a teaching tool to enhance the learning of many biological concepts from genetics, development, and behavior to the understanding of the local watershed. Traditionally, in both research and teaching, zebrafish work has focused on embryonic stages; however, later stages, from larval through adulthood, are increasingly being examined. Defining developmental stages based on age is a problematic way to assess maturity, because many environmental factors, such as temperature, population density, and water quality, impact growth and maturation. Fish length and characterization of key external morphological traits are considered better markers for maturation state. While a number of staging series exist for zebrafish, here we present a simplified normalization table of post-embryonic maturation well suited to both educational and research use. Specifically, we utilize fish size and four easily identified external morphological traits (pigment pattern, tail fin, anal fin, and dorsal fin morphology) to describe three larval stages, a juvenile stage, and an adult stage. These simplified maturation standards will be a useful tool for both educational and research protocols. PMID:24979389

  12. Neuroprotective Role of the PI3 Kinase/Akt Signaling Pathway in Zebrafish

    PubMed Central

    Chen, Shuang; Liu, Yunzhang; Rong, Xiaozhi; Li, Yun; Zhou, Jianfeng; Lu, Ling

    2017-01-01

    Neuronal survival and growth in the embryo is controlled partly by trophic factors. For most trophic factors (such as Insulin-like growth factor-1), the ability to regulate cell survival has been attributed to the phosphoinositide 3-kinase (PI3K)/Akt kinase cascade. This study presents data illustrating the role of PI3K/Akt in attainment of normal brain size during zebrafish embryogenesis. Blocking PI3K with inhibitor LY294002 caused a significant reduction in brain size (in addition to global growth retardation) during zebrafish embryogenesis. This PI3 Kinase inhibition-induced brain size decrease was recovered by the overexpression of myristoylated Akt (myr-Akt), a constitutive form of Akt. Further analysis reveals that expressing exogenous myr-Akt significantly augmented brain size. Whole mount in situ hybridization analysis of several marker genes showed that myr-Akt overexpression did not alter brain patterning. Furthermore, the expression of myr-Akt was found to protect neuronal cells from apoptosis induced by heat shock and UV light, suggesting that inhibition of neuronal cell death may be part of the underlying cause of the increased brain size. These data provide a foundation for addressing the role of PI3K/Akt in brain growth during zebrafish embryogenesis. PMID:28228749

  13. Loss of col8a1a Function during Zebrafish Embryogenesis Results in Congenital Vertebral Malformations

    PubMed Central

    Gray, Ryan S.; Wilm, Thomas; Smith, Jeff; Bagnat, Michel; Dale, Rodney M.; Topczewski, Jacek; Johnson, Stephen L.; Solnica-Krezel, Lilianna

    2014-01-01

    Congenital vertebral malformations (CVM) occur in 1 in 1,000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles (m531, vu41, vu105) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue. PMID:24333517

  14. Amigo Adhesion Protein Regulates Development of Neural Circuits in Zebrafish Brain*

    PubMed Central

    Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A.; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

    2014-01-01

    The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. PMID:24904058

  15. Zebrafish ambra1a and ambra1b Knockdown Impairs Skeletal Muscle Development

    PubMed Central

    Skobo, Tatjana; Benato, Francesca; Grumati, Paolo; Meneghetti, Giacomo; Cianfanelli, Valentina; Castagnaro, Silvia; Chrisam, Martina; Di Bartolomeo, Sabrina; Bonaldo, Paolo; Cecconi, Francesco; Valle, Luisa Dalla

    2014-01-01

    The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle. PMID:24922546

  16. The N-terminal acetyltransferase Naa10 is essential for zebrafish development

    PubMed Central

    Ree, Rasmus; Myklebust, Line M.; Thiel, Puja; Foyn, Håvard; Fladmark, Kari E.; Arnesen, Thomas

    2015-01-01

    N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish. PMID:26251455

  17. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

    Petrie-Hanson, Lora; Hohn, Claudia; Hanson, Larry

    2009-01-01

    Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

  18. What optimization principle explains the zebrafish vasculature?

    NASA Astrophysics Data System (ADS)

    Chang, Shyr-Shea; Baek, Kyung In; Hsiai, Tzung; Roper, Marcus

    2016-11-01

    Many multicellular organisms depend on biological transport networks; from the veins of leaves to the animal circulatory system, to redistribute nutrients internally. Since natural selection rewards efficiency, those networks are thought to minimize the cost of maintaining the flow inside. But optimizing these costs creates tradeoffs with other functions, e.g. mixing or uniform distribution of nutrients. We develop an extended Lagrange multiplier approach that allows the optimization of general network functionals. We also follow the real zebrafish vasculature and blood flows during organism development. Taken together, our work shows that the challenge of uniform oxygen perfusion, and not transport efficiency, explain zebrafish vascular organization. Ruth L. Kirschstein National Research Service Award (T32-GM008185).

  19. Parallel mechanisms for visual search in zebrafish.

    PubMed

    Proulx, Michael J; Parker, Matthew O; Tahir, Yasser; Brennan, Caroline H

    2014-01-01

    Parallel visual search mechanisms have been reported previously only in mammals and birds, and not animals lacking an expanded telencephalon such as bees. Here we report the first evidence for parallel visual search in fish using a choice task where the fish had to find a target amongst an increasing number of distractors. Following two-choice discrimination training, zebrafish were presented with the original stimulus within an increasing array of distractor stimuli. We found that zebrafish exhibit no significant change in accuracy and approach latency as the number of distractors increased, providing evidence of parallel processing. This evidence challenges theories of vertebrate neural architecture and the importance of an expanded telencephalon for the evolution of executive function.

  20. Attraction rules: germ cell migration in zebrafish.

    PubMed

    Raz, Erez; Reichman-Fried, Michal

    2006-08-01

    The migration of zebrafish primordial germ cell towards the region where the gonad develops is guided by the chemokine SDF-1a. Recent studies show that soon after their specification, the cells undergo a series of morphological alterations before they become motile and are able to respond to attractive cues. As migratory cells, primordial germ cells move towards their target while correcting their path upon exiting a cyclic phase in which morphological cell polarity is lost. In the following stages, the cells gather at specific locations and move as cell clusters towards their final target. In all of these stages, zebrafish germ cells respond as individual cells to alterations in the shape of the sdf-1a expression domain, by directed migration towards their target - the position where the gonad develops.

  1. Understanding cardiac sarcomere assembly with zebrafish genetics.

    PubMed

    Yang, Jingchun; Shih, Yu-Huan; Xu, Xiaolei

    2014-09-01

    Mutations in sarcomere genes have been found in many inheritable human diseases, including hypertrophic cardiomyopathy. Elucidating the molecular mechanisms of sarcomere assembly shall facilitate understanding of the pathogenesis of sarcomere-based cardiac disease. Recently, biochemical and genomic studies have identified many new genes encoding proteins that localize to the sarcomere. However, their precise functions in sarcomere assembly and sarcomere-based cardiac disease are unknown. Here, we review zebrafish as an emerging vertebrate model for these studies. We summarize the techniques offered by this animal model to manipulate genes of interest, annotate gene expression, and describe the resulting phenotypes. We survey the sarcomere genes that have been investigated in zebrafish and discuss the potential of applying this in vivo model for larger-scale genetic studies.

  2. Osteogenic programs during zebrafish fin regeneration

    PubMed Central

    Watson, Claire J; Kwon, Ronald Y

    2015-01-01

    Recent advances in genomic, screening and imaging technologies have provided new opportunities to examine the molecular and cellular landscape underlying human physiology and disease. In the context of skeletal research, technologies for systems genetics, high-throughput screening and high-content imaging can aid an unbiased approach when searching for new biological, pathological or therapeutic pathways. However, these approaches necessitate the use of specialized model systems that rapidly produce a phenotype, are easy to manipulate, and amenable to optical study, all while representing mammalian bone physiologies at the molecular and cellular levels. The emerging use of zebrafish (Danio rerio) for modeling human disease highlights its potential to accelerate therapeutic and pathway discovery in the mammalian skeleton. In this review, we consider the potential value of zebrafish fin ray regeneration (a rapid, genetically tractable and optically transparent model of intramembranous ossification) as a translational model for such studies. PMID:26421148

  3. The Morphogenesis of Cranial Sutures in Zebrafish

    PubMed Central

    Topczewska, Jolanta M.; Shoela, Ramy A.; Tomaszewski, Joanna P.; Mirmira, Rupa B.; Gosain, Arun K.

    2016-01-01

    Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births. PMID:27829009

  4. Dynamics of DNA hydroxymethylation in zebrafish.

    PubMed

    Kamstra, Jorke H; Løken, Marianne; Aleström, Peter; Legler, Juliette

    2015-06-01

    During embryonic development in mammals, most of the methylated cytosines in the paternal genome are converted to 5-hydroxymethyldeoxycytidine (hmC), as part of DNA methylation reprogramming. Recent data also suggest tissue-specific functional roles of hmC, perhaps as an epigenetic mark. However, limited data are available on the levels and tissue distribution in zebrafish. In this study, we used high-performance liquid chromatography mass spectrometry to quantify hmC and 5-methyldeoxycytidine (mC) in zebrafish during development and in different tissues of the adult fish. Low levels of mC were found at 0.5 hours postfertilization (hpf) (1-2 cell stage) (1.9%), and increased to 8.4% by 96 hpf, with similar levels observed in different adult tissues. No hmC was detected up to 12 hpf, but levels increased during development from 24 up to 96 hpf (0.23%). In tissues, the highest levels of hmC were found in the brain (0.49%), intermediate levels in muscle (0.13%), liver (0.08%), and intestine (0.06%) and low levels in testis (0.01%), with an inverse correlation between hmC and mC. Our results indicate similar tissue distribution and levels of hmC between zebrafish and mammals, but distinct differences during embryonic development. Although more research is needed, these results support the use of zebrafish as an alternative model in the elucidation of tissue-specific functions of hmC.

  5. BDE 49 and developmental toxicity in zebrafish

    PubMed Central

    McClain, Valerie; Stapleton, Heather M.; Gallagher, Evan

    2011-01-01

    The polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants. Human health concerns of these agents have largely centered upon their potential to elicit reproductive and developmental effects. Of the various congeners, BDE 49 (2,2’,4,5’-tetrabromodiphenyl ether) has been poorly studied, despite the fact that it is often detected in the tissues of fish and wildlife species. Furthermore, we have previously shown that BDE 49 is a metabolic debromination product of BDE 99 hepatic metabolism in salmon, carp and trout, underscoring the need for a better understanding of biological effects. In the current study, we investigated the developmental toxicity of BDE 49 using the zebrafish (Danio rerio) embryo larval model. Embryo and larval zebrafish were exposed to BDE 49 at either 5 hours post fertilization (hpf) or 24 hpf and monitored for developmental and neurotoxicity. Exposure to BDE 49 at concentrations of 4 µM- 32 µM caused a dose-dependent loss in survivorship at 6 days post fertilization (dpf). Morphological impairments were observed prior to the onset of mortality, the most striking of which included severe dorsal curvatures of the tail. The incidence of dorsal tail curvatures was dose and time dependent. Exposure to BDE 49 caused cardiac toxicity as evidenced by a significant reduction in zebrafish heart rates at 6 dpf but not earlier, suggesting that cardiac toxicity was non-specific and associated with physiological stress. Neurobehavioral injury from BDE 49 was evidenced by an impairment of touch-escape responses observed at 5 dpf. Our results indicate that BDE 49 is a developmental toxicant in larval zebrafish that can cause morphological abnormalities and adversely affect neurobehavior. The observed toxicities from BDE 49 were similar in scope to those previously reported for the more common tetrabrominated congener, BDE 47, and also for other lower brominated PBDEs, suggest that these compounds may share similarities in risk to

  6. TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

    NASA Astrophysics Data System (ADS)

    Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

    2016-02-01

    Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle.

  7. Acid-sensing ion channels (ASICs) 2 and 4.2 are expressed in the retina of the adult zebrafish.

    PubMed

    Viña, E; Parisi, V; Sánchez-Ramos, C; Cabo, R; Guerrera, M C; Quirós, L M; Germanà, A; Vega, J A; García-Suárez, O

    2015-05-01

    Acid-sensing ion channels (ASICs) are H(+)-gated, voltage-insensitive cation channels involved in synaptic transmission, mechanosensation and nociception. Different ASICs have been detected in the retina of mammals but it is not known whether they are expressed in adult zebrafish, a commonly used animal model to study the retina in both normal and pathological conditions. We study the expression and distribution of ASIC2 and ASIC4 in the retina of adult zebrafish and its regulation by light using PCR, in situ hybridization, western blot and immunohistochemistry. We detected mRNA encoding zASIC2 and zASIC4.2 but not zASIC4.1. ASIC2, at the mRNA or protein level, was detected in the outer nuclear layer, the outer plexiform layer, the inner plexiform layer, the retinal ganglion cell layer and the optic nerve. ASIC4 was expressed in the photoreceptors layer and to a lesser extent in the retinal ganglion cell layer. Furthermore, the expression of both ASIC2 and ASIC4.2 was down-regulated by light and darkness. These results are the first demonstration that ASIC2 and ASIC4 are expressed in the adult zebrafish retina and suggest that zebrafish could be used as a model organism for studying retinal pathologies involving ASICs.

  8. TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

    PubMed Central

    Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

    2016-01-01

    Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle. PMID:26898711

  9. Heat-induced masculinization in domesticated zebrafish is family-specific and yields a set of different gonadal transcriptomes.

    PubMed

    Ribas, Laia; Liew, Woei Chang; Díaz, Noèlia; Sreenivasan, Rajini; Orbán, László; Piferrer, Francesc

    2017-02-07

    Understanding environmental influences on sex ratios is important for the study of the evolution of sex-determining mechanisms and for evaluating the effects of global warming and chemical pollution. Fishes exhibit sexual plasticity, but the underlying mechanisms of environmental effects on their reproduction are unclear even in the well-established teleost research model, the zebrafish. Here we established the conditions to study the effects of elevated temperature on zebrafish sex. We showed that sex ratio response to elevated temperature is family-specific and typically leads to masculinization (female-to-male sex reversal), resulting in neomales. These results uncovered genotype-by-environment interactions that support a polygenic sex determination system in domesticated (laboratory) zebrafish. We found that some heat-treated fish had gene expression profiles similar to untreated controls of the same sex, indicating that they were resistant to thermal effects. Further, most neomales had gonadal transcriptomes similar to that of regular males. Strikingly, we discovered heat-treated females that displayed a normal ovarian phenotype but with a "male-like" gonadal transcriptome. Such major transcriptomic reprogramming with preserved organ structure has never been reported. Juveniles were also found to have a male-like transcriptome shortly after exposure to heat. These findings were validated by analyzing the expression of genes and signaling pathways associated with sex differentiation. Our results revealed a lasting thermal effect on zebrafish gonads, suggesting new avenues for detection of functional consequences of elevated temperature in natural fish populations in a global warming scenario.

  10. Normal clotting.

    PubMed

    Moran, Theresa A; Viele, Carol S

    2005-11-01

    To review the normal coagulation process and the mechanisms that lead to abnormal clotting. Primary and tertiary literature and the authors' clinical experience. The process of coagulation is complex and can be easily misunderstood. It is important to be familiar with normal coagulation before one can comprehend the coagulopathies associated with malignancies. A thorough understanding of the coagulation process is a critical prerequisite to caring for patients with clotting disorders. Once the normal clotting process is understood, the abnormal becomes easier to recognize and the cancer-associated dysfunctions more readily identified.

  11. Hyperglycemia induces memory impairment linked to increased acetylcholinesterase activity in zebrafish (Danio rerio).

    PubMed

    Capiotti, Katiucia Marques; De Moraes, Daiani Almeida; Menezes, Fabiano Peres; Kist, Luiza Wilges; Bogo, Maurício Reis; Da Silva, Rosane Souza

    2014-11-01

    Diabetes mellitus, which causes hyperglycemia, affects the central nervous system and can impairs cognitive functions, such as memory. The aim of this study was to investigate the effects of hyperglycemia on memory as well as on the activity of acethylcholinesterase. Hyperglycemia was induced in adult zebrafish by immersion in glucose 111mM by 14 days. The animals were divided in 4 groups: control, glucose-treated, glucose-washout 7-days and glucose-washout 14-days. We evaluated the performance in inhibitory avoidance task and locomotor activity. We also determined acethylcholinesterase activity and gene expression from whole brain. In order to counteract the effect of hyperglycemia underlined by effects on acethylcholinesterase activity, we treated the animals with galantamine (0.05ng/g), an inhibitor of this enzyme. Also we evaluated the gene expression of insulin receptor and glucose transporter from zebrafish brain. The hyperglycemia promoted memory deficit in adult zebrafish, which can be explained by increased AChE activity. The ache mRNA levels from zebrafish brain were decrease in 111mM glucose group and returned to normal levels after 7 days of glucose withdrawal. Insulin receptors (insra-1, insra-2, insrb-1 and insrb-2) and glut-3 mRNA levels were not significantly changed. Our results also demonstrated that galantamine was able to reverse the memory deficit caused by hyperglycemia, demonstrating that these effects involve modulation of AChE activity. These data suggest that the memory impairment induced by hyperglycemia is underlined by the cholinergic dysfunction caused by the mechanisms involving the control of acetylcholinesterase function and gene expression. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification

    PubMed Central

    Cacialli, Pietro; Gueguen, Marie-Madeleine; Coumailleau, Pascal; D’Angelo, Livia; Kah, Olivier; Lucini, Carla; Pellegrini, Elisabeth

    2016-01-01

    Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations. PMID:27336917

  13. Effects of simulated-microgravity on zebrafish embryonic development and microRNA expression

    NASA Astrophysics Data System (ADS)

    Hang, Xiaoming; Sun, Yeqing; Zhang, Meng; Li, Hui

    2012-07-01

    Microgravity is a constant physical factor astronauts must meet during space flight. Therefore, the mechanism of microgravity-induced biological effects is one of the most important issues in space biological studies. In this research, zebrafish (Danio rerio) embryos at different development stages were exposed to simulated microgravity, respectively, using a rotary cell culture system (RCCS) designed by NASA. Biological effects of simulated microgravity on zebrafish embryos were investigated at the phenotypic and microRNA expression levels. Malformation rate and mortality rate were found increased after simulated microgravity exposure. Body length and heart rate were also increased during microgravity exposure and after a shot period of gravity recovery, but both returned to normal level after 10 days and 7 days of gravity recovery, respectively. Additionally, significant changes in microRNA expression profiles of zebrafish embryos were observed, depending on the development stages of embyos exposed to simulated microgravity and the exposure time. All together, nine miRNAs showed significant changes after three different microgravity exposures (8-72hpf, 24-72hpf and 24-48hpf). Four miRNAs, dre-miR-738, dre-miR-133a, dre-miR-133b and dre-miR-22a, were up-regulated. Two miRNAs, dre-miR-1 and dre-miR-16a, were down-regulated. The other three miRNAs, dre-miR-204, dre-miR-9* and dre-miR-429, were found up-regulated when microgravity exposures ended at 72hpf, but down-regulated when microgravity exposures ended at 48hpf. Above results demonstrated microRNA expression of zebrafish embryos could be induced by both embryonic development stage and simulated microgravity. Key Words: Danio rerio; Simulated-microgravity; embryonic devlopment; microRNA expression

  14. Pathogen invasion changes the intestinal microbiota composition and induces innate immune responses in the zebrafish intestine.

    PubMed

    Yang, Hui-Ting; Zou, Song-Song; Zhai, Li-Juan; Wang, Yao; Zhang, Fu-Miao; An, Li-Guo; Yang, Gui-Wen

    2017-09-28

    Numerous bacteria are harbored in the animal digestive tract and are impacted by several factors. Intestinal microbiota homeostasis is critical for maintaining the health of an organism. However, how pathogen invasion affects the microbiota composition has not been fully clarified. The mechanisms for preventing invasion by pathogenic microorganisms are yet to be elucidated. Zebrafish is a useful model for developmental biology, and studies in this organism have gradually become focused on intestinal immunity. In this study, we analyzed the microbiota of normal cultivated and infected zebrafish intestines, the aquarium water and feed samples. We found that the predominant bacteria in the zebrafish intestine belonged to Gammaproteobacteria (67%) and that feed and environment merely influenced intestinal microbiota composition only partially. Intestinal microbiota changed after a pathogenic bacterial challenge. At the genus level, the abundance of some pathogenic intestinal bacteria increased, and these genera included Halomonas (50%), Pelagibacterium (3.6%), Aeromonas (2.6%), Nesterenkonia (1%), Chryseobacterium (3.4‰), Mesorhizobium (1.4‰), Vibrio (1‰), Mycoplasma (0.7‰) and Methylobacterium (0.6‰) in IAh group. However, the abundance of some beneficial intestinal bacteria decreased, and these genera included Nitratireductor (0.8‰), Enterococcus (0.8‰), Brevundimonas (0.7‰), Lactococcus (0.7‰) and Lactobacillus (0.4‰). Additionally, we investigated the innate immune responses after infection. ROS levels in intestine increased in the early stages after a challenge and recovered subsequently. The mRNA levels of antimicrobial peptide genes lectin, hepcidin and defensin1, were upregulated in the intestine after pathogen infection. These results suggested that the invasion of pathogen could change the intestinal microbiota composition and induce intestinal innate immune responses in zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Insulin-like growth factor-2 regulates early neural and cardiovascular system development in zebrafish embryos.

    PubMed

    Hartnett, Lori; Glynn, Catherine; Nolan, Catherine M; Grealy, Maura; Byrnes, Lucy

    2010-01-01

    The insulin-like growth factor (IGF) family is essential for normal embryonic growth and development and it is highly conserved through vertebrate evolution. However, the roles that the individual members of the IGF family play in embryonic development have not been fully elucidated. This study focuses on the role of IGF-2 in zebrafish embryonic development. Two igf-2 genes, igf-2a and igf-2b, are present in the zebrafish genome. Antisense morpholinos were designed to knock down both igf-2 genes. The neural and cardiovascular defects in IGF-2 morphant embryos were then examined further using wholemount in situ hybridisation, TUNEL analysis and O-dianisidine staining. Knockdown of igf-2a or igf-2b resulted in ventralised embryos with reduced growth, reduced eyes, disrupted brain structures and a disrupted cardiovascular system, with igf-2b playing a more significant role in development. During gastrulation, igf-2a and igf-2b are required for development of anterior neural structures and for regulation of genes critical to dorsal-ventral patterning. As development proceeds, igf-2a and igf-2b play anti-apoptotic roles. Gene expression analysis demonstrates that igf-2a and igf-2b play overlapping roles in angiogenesis and cardiac outflow tract development. Igf-2b is specifically required for cardiac valve development and cardiac looping. Injection of a dominant negative IGF-1 receptor led to similar defects in angiogenesis and cardiac valve development, indicating IGF-2 signals through this receptor to regulate cardiovascular development. This is the first study describing two functional igf-2 genes in zebrafish. This work demonstrates that igf-2a and igf-2b are critical to neural and cardiovascular development in zebrafish embryos. The finding that igf-2a and igf-2b do not act exclusively in a redundant manner may explain why both genes have been stably maintained in the genome.

  16. Standardized Welfare Terms for the Zebrafish Community

    PubMed Central

    Karp, Natasha A.; Blackledge, Samuel; Clark, Bradley; Keeble, Rosemary; Kovacs, Ceri; Murray, Katrina N.; Price, Michael; Thompson, Peter; Bussell, James

    2016-01-01

    Abstract Managing the welfare of laboratory animals is critical to animal health, vital in the understanding of phenotypes created by treatment or genetic alteration and ensures compliance of regulations. Part of an animal welfare assessment is the requirement to record observations, ensuring all those responsible for the animals are aware of their health status and can act accordingly. Although the use of zebrafish in research continues to increase, guidelines for conducting welfare assessments and the reporting of observations are considered unclear compared to mammalian species. To support the movement of zebrafish between facilities, significant improvement would be achieved through the use of standardized terms to ensure clarity and consistency between facilities. Improving the clarity of terminology around welfare not only addresses our ethical obligation but also supports the research goals and provides a searchable description of the phenotypes. A Collaboration between the Wellcome Trust Sanger Institute and Cambridge University (Department of Medicine-Laboratory of Molecular Biology) has led to the creation of the zebrafish welfare terms from which standardization of terminology can be achieved. PMID:27096380

  17. Single stimulus learning in zebrafish larvae

    PubMed Central

    O’Neale, Ashley; Ellis, Joseph; Creton, Robbert; Colwill, Ruth M.

    2014-01-01

    Learning about a moving visual stimulus was examined in zebrafish larvae using an automated imaging system and a t1-t2 design. In three experiments, zebrafish larvae were exposed to one of two inputs at t1 (either a gray bouncing disk or an identical but stationary disk) followed by a common test at t2 (the gray bouncing disk). Using 7 days post-fertilization (dpf) larvae and 12 stimulus exposures, Experiment 1 established that these different treatments produced differential responding to the moving disk during testing. Larvae familiar with the moving test stimulus were significantly less likely to be still in its presence than larvae that had been exposed to the identical but stationary stimulus. Experiment 2 confirmed this result in 7 dpf larvae and extended the finding to 5 and 6 dpf larvae. Experiment 3 found differential responding to the moving test stimulus with 4 or 8 stimulus exposures but not with just one exposure in 7 dpf larvae. These results provide evidence for learning in very young zebrafish larvae. The merits and challenges of the t1-t2 framework to study learning are discussed. PMID:24012906

  18. Short stories on zebrafish long noncoding RNAs.

    PubMed

    Haque, Shadabul; Kaushik, Kriti; Leonard, Vincent Elvin; Kapoor, Shruti; Sivadas, Ambily; Joshi, Adita; Scaria, Vinod; Sivasubbu, Sridhar

    2014-12-01

    The recent re-annotation of the transcriptome of human and other model organisms, using next-generation sequencing approaches, has unravelled a hitherto unknown repertoire of transcripts that do not have a potential to code for proteins. These transcripts have been largely classified into an amorphous class popularly known as long noncoding RNAs (lncRNA). This discovery of lncRNAs in human and other model systems have added a new layer to the understanding of gene regulation at the transcriptional and post-transcriptional levels. In recent years, three independent studies have discovered a number of lncRNAs expressed in different stages of zebrafish development and adult tissues using a high-throughput RNA sequencing approach, significantly adding to the repertoire of genes known in zebrafish. A subset of these transcripts also shows distinct and specific spatiotemporal patterns of gene expression, pointing to a tight regulatory control and potential functional roles in development, organogenesis, and/ or homeostasis. This review provides an overview of the lncRNAs in zebrafish and discusses how their discovery could provide new insights into understanding biology, explaining mutant phenotypes, and helping in potentially modeling disease processes.

  19. Standardized Welfare Terms for the Zebrafish Community.

    PubMed

    Goodwin, Nicola; Karp, Natasha A; Blackledge, Samuel; Clark, Bradley; Keeble, Rosemary; Kovacs, Ceri; Murray, Katrina N; Price, Michael; Thompson, Peter; Bussell, James

    2016-07-01

    Managing the welfare of laboratory animals is critical to animal health, vital in the understanding of phenotypes created by treatment or genetic alteration and ensures compliance of regulations. Part of an animal welfare assessment is the requirement to record observations, ensuring all those responsible for the animals are aware of their health status and can act accordingly. Although the use of zebrafish in research continues to increase, guidelines for conducting welfare assessments and the reporting of observations are considered unclear compared to mammalian species. To support the movement of zebrafish between facilities, significant improvement would be achieved through the use of standardized terms to ensure clarity and consistency between facilities. Improving the clarity of terminology around welfare not only addresses our ethical obligation but also supports the research goals and provides a searchable description of the phenotypes. A Collaboration between the Wellcome Trust Sanger Institute and Cambridge University (Department of Medicine-Laboratory of Molecular Biology) has led to the creation of the zebrafish welfare terms from which standardization of terminology can be achieved.

  20. Social dominance modulates eavesdropping in zebrafish

    PubMed Central

    Abril-de-Abreu, Rodrigo; Cruz, Ana S.; Oliveira, Rui F.

    2015-01-01

    Group living animals may eavesdrop on signalling interactions between conspecifics and integrate it with their own past social experience in order to optimize the use of relevant information from others. However, little is known about this interplay between public (eavesdropped) and private social information. To investigate it, we first manipulated the dominance status of bystander zebrafish. Next, we either allowed or prevented bystanders from observing a fight. Finally, we assessed their behaviour towards the winners and losers of the interaction, using a custom-made video-tracking system and directional analysis. We found that only dominant bystanders who had seen the fight revealed a significant increase in directional focus (a measure of attention) towards the losers of the fights. Furthermore, our results indicate that information about the fighters' acquired status was collected from the signalling interaction itself and not from post-interaction status cues, which implies the existence of individual recognition in zebrafish. Thus, we show for the first time that zebrafish, a highly social model organism, eavesdrop on conspecific agonistic interactions and that this process is modulated by the eavesdroppers' dominance status. We suggest that this type of integration of public and private information may be ubiquitous in social learning processes. PMID:26361550

  1. The zebrafish infraorbital bones: a descriptive study.

    PubMed

    Chang, Carolyn; Franz-Odendaal, Tamara Anne

    2014-02-01

    The infraorbital (IO) bone series, a component of the circumorbital series, makes up five of the eight dermal bones found in the orbital region of the zebrafish skull. Ossifying in a set sequence, the IOs are closely associated with the cranial lateral line system as they house neuromast sensory receptors in bony canals. We conducted a detailed analysis of the condensation to mineralization phases of development of these bones. Our analyses involved both bone and osteoblast staining of zebrafish at 20 different time points. IO bone condensations are shaped as templates for the final bone shape, and they mineralize at one or more centers of ossification. Initially, mineralization is closely associated with the lateral line canals and/or foramen, and the onset of mineralization is temporally variable. Canal wall mineralization is a process that continues into adulthood and completely mineralized canal roofs were not found. Our comprehensive growth series detailing the ossification of each IO bone provides important insight into the growth and development of this series of neural crest-derived flat bones in the zebrafish craniofacial skeleton.

  2. Phenylthiourea disrupts thyroid function in developing zebrafish.

    PubMed

    Elsalini, Osama A; Rohr, Klaus B

    2003-01-01

    Thyroid hormone (T4) can be detected in thyroid follicles in wild-type zebrafish larvae from 3 days of development, when the thyroid has differentiated. In contrast, embryos or larvae treated with goitrogens (substances such as methimazole, potassium percholorate, and 6-n-propyl-2-thiouracil) are devoid of thyroid hormone immunoreactivity. Phenythiourea (PTurea; also commonly known as PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. PTurea contains a thiocarbamide group that is responsible for goitrogenic activity in methimazole and 6-n-propyl-2-thiouracil. In the present study, we show that commonly used doses of 0.003% PTurea abolish T4 immunoreactivity of the thyroid follicles of zebrafish larvae. As development of the thyroid gland is not affected, these data suggest that PTurea blocks thyroid hormone production. Like other goitrogens, PTurea causes delayed hatching, retardation and malformation of embryos or larvae with increasing doses. At doses of 0.003% PTurea, however, toxic side effects seem to be at a minimum, and the maternal contribution of the hormone might compensate for compromised thyroid function during the first days of development.

  3. Transgenerational analysis of transcriptional silencing in zebrafish

    PubMed Central

    Akitake, Courtney M.; Macurak, Michelle; Halpern, Marnie E.; Goll, Mary G.

    2011-01-01

    The yeast Gal4/UAS transcriptional activation system is a powerful tool for regulating gene expression in Drosophila and has been increasing in popularity for developmental studies in zebrafish. It is also useful for studying the basis of de novo transcriptional silencing. Fluorescent reporter genes under the control of multiple tandem copies of the upstream activator sequence (UAS) often show evidence of variegated expression and DNA methylation in transgenic zebrafish embryos. To characterize this systematically, we monitored the progression of transcriptional silencing of UAS-regulated transgenes that differ in their integration sites and in the repetitive nature of the UAS. Transgenic larvae were examined in three generations for tissue-specific expression of a green fluorescent protein (GFP) reporter and DNA methylation at the UAS. Single insertions containing four distinct upstream activator sequences were far less susceptible to methylation than insertions containing fourteen copies of the same UAS. In addition, transgenes that integrated in or adjacent to transposon sequence exhibited silencing regardless of the number of UAS sites included in the transgene. Placement of promoter-driven Gal4 upstream of UAS-regulated responder genes in a single bicistronic construct also appeared to accelerate silencing and methylation. The results demonstrate the utility of the zebrafish for efficient tracking of gene silencing mechanisms across several generations, as well as provide useful guidelines for optimal Gal4-regulated gene expression in organisms subject to DNA methylation. PMID:21223961

  4. Zebrafish Models for Dyslipidemia and Atherosclerosis Research

    PubMed Central

    Schlegel, Amnon

    2016-01-01

    Atherosclerotic cardiovascular disease is the leading cause of death. Elevated circulating concentrations of lipids are a central pathogenetic driver of atherosclerosis. While numerous effective therapies for this condition have been developed, there is substantial unmet need for this pandemic illness. Here, I will review nutritional, physiological, genetic, and pathological discoveries in the emerging zebrafish model for studying dyslipidemia and atherosclerosis. The technical and physiological advantages and the pharmacological potential of this organism for discovery and validation of dyslipidemia and atherosclerosis targets are stressed through summary of recent findings. An emerging literature shows that zebrafish, through retention of a cetp ortholog gene and high sensitivity to ingestion of excess cholesterol, rapidly develops hypercholesterolemia, with a pattern of distribution of lipid species in lipoprotein particles similar to humans. Furthermore, recent studies leveraging the optical transparency of zebrafish larvae to monitor the fate of these ingested lipids have provided exciting insights to the development of dyslipidemia and atherosclerosis. Future directions for investigation are considered, with particular attention to the potential for in vivo cell biological study of atherosclerotic plaques. PMID:28018294

  5. Short Stories on Zebrafish Long Noncoding RNAs

    PubMed Central

    Haque, Shadabul; Kaushik, Kriti; Leonard, Vincent Elvin; Kapoor, Shruti; Sivadas, Ambily; Joshi, Adita

    2014-01-01

    Abstract The recent re-annotation of the transcriptome of human and other model organisms, using next-generation sequencing approaches, has unravelled a hitherto unknown repertoire of transcripts that do not have a potential to code for proteins. These transcripts have been largely classified into an amorphous class popularly known as long noncoding RNAs (lncRNA). This discovery of lncRNAs in human and other model systems have added a new layer to the understanding of gene regulation at the transcriptional and post-transcriptional levels. In recent years, three independent studies have discovered a number of lncRNAs expressed in different stages of zebrafish development and adult tissues using a high-throughput RNA sequencing approach, significantly adding to the repertoire of genes known in zebrafish. A subset of these transcripts also shows distinct and specific spatiotemporal patterns of gene expression, pointing to a tight regulatory control and potential functional roles in development, organogenesis, and/ or homeostasis. This review provides an overview of the lncRNAs in zebrafish and discusses how their discovery could provide new insights into understanding biology, explaining mutant phenotypes, and helping in potentially modeling disease processes. PMID:25110965

  6. Characterization of the Enigma family in zebrafish.

    PubMed

    Ott, Elisabeth B; Sakalis, Philippe A; Marques, Ines J; Bagowski, Christoph P

    2007-11-01

    The three Enigma subfamily proteins, Enigma, Enigma homologue, and Cypher/ZASP belong to the PDZ and LIM encoding protein family, which is characterized by the presence of a PDZ- and one or more LIM domains. PDZ/LIM proteins play important biological roles, and all members have been shown to associate with the actin cytoskeleton. We describe here the splice form specific expression patterns for the three Enigma subfamily members during zebrafish embryogenesis. Whole-mount in situ hybridization revealed common and distinct expression patterns for the different PDZ or LIM domain encoding splice variants. We further studied the role of enigma in zebrafish development. Enigma knockdown appeared to be embryonic lethal shortly after the end of gastrulation and in few surviving embryos led to elongation defects and disorganized somites. In summary, we show here the temporal and spatial expression patterns of the three Enigma family members and their PDZ and LIM domain encoding splice forms during zebrafish embryogenesis. Our results suggest that enigma is important for the formation and organization of somites and might play an important role for actin cytoskeleton organization during development.

  7. Repressor Dimerization in the Zebrafish Somitogenesis Clock

    PubMed Central

    Cinquin, Olivier

    2007-01-01

    The oscillations of the somitogenesis clock are linked to the fundamental process of vertebrate embryo segmentation, yet little is known about their generation. In zebrafish, it has been proposed that Her proteins repress the transcription of their own mRNA. However, in its simplest form, this model is incompatible with the fact that morpholino knockdown of Her proteins can impair expression of their mRNA. Simple self-repression models also do not account for the spatiotemporal pattern of gene expression, with waves of gene expression shrinking as they propagate. Here we study computationally the networks generated by the wealth of dimerization possibilities amongst transcriptional repressors in the zebrafish somitogenesis clock. These networks can reproduce knockdown phenotypes, and strongly suggest the existence of a Her1–Her7 heterodimer, so far untested experimentally. The networks are the first reported to reproduce the spatiotemporal pattern of the zebrafish somitogenesis clock; they shed new light on the role of Her13.2, the only known link between the somitogenesis clock and positional information in the paraxial mesoderm. The networks can also account for perturbations of the clock by manipulation of FGF signaling. Achieving an understanding of the interplay between clock oscillations and positional information is a crucial first step in the investigation of the segmentation mechanism. PMID:17305423

  8. Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

    PubMed Central

    Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

    2013-01-01

    Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

  9. Characterization of behavioral and endocrine effects of LSD on zebrafish.

    PubMed

    Grossman, Leah; Utterback, Eli; Stewart, Adam; Gaikwad, Siddharth; Chung, Kyung Min; Suciu, Christopher; Wong, Keith; Elegante, Marco; Elkhayat, Salem; Tan, Julia; Gilder, Thomas; Wu, Nadine; Dileo, John; Cachat, Jonathan; Kalueff, Allan V

    2010-12-25

    Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  10. A bioenergetic model for zebrafish Danio rerio (Hamilton)

    USGS Publications Warehouse

    Chizinski, C.J.; Sharma, Bibek; Pope, K.L.; Patino, R.

    2008-01-01

    A bioenergetics model was developed from observed consumption, respiration and growth rates for zebrafish Danio rerio across a range (18-32?? C) of water temperatures, and evaluated with a 50 day laboratory trial at 28?? C. No significant bias in variable estimates was found during the validation trial; namely, predicted zebrafish mass generally agreed with observed mass. ?? 2008 The Authors.

  11. Identifying Structural Alerts Based on Zebrafish Developmental Morphological Toxicity (TDS)

    EPA Science Inventory

    Zebrafish constitute a powerful alternative animal model for chemical hazard evaluation. To provide an in vivo complement to high-throughput screening data from the ToxCast program, zebrafish developmental toxicity screens were conducted on the ToxCast Phase I (Padilla et al., 20...

  12. Using Zebrafish to Study Kidney Development and Disease.

    PubMed

    Jerman, Stephanie; Sun, Zhaoxia

    2017-01-01

    The kidneys are a crucial pair of organs that are responsible for filtering the blood to remove waste, maintain electrolyte and water homeostasis, and regulate blood pressure. There are a number of factors, both genetic and environmental, that can impair the function of the kidneys resulting in significant morbidity and mortality for millions of people affected by kidney disease worldwide. The zebrafish, Danio rerio, has emerged as an attractive vertebrate model in the study of kidney development and disease and has proven to be a powerful tool in the advancement of how kidney development occurs in vertebrates and how the kidney repairs itself after injury. Zebrafish share significant similarities in kidney development and composition of nephrons, the functional unit of the kidney. This makes the zebrafish a very promising model to study the mechanisms by which renal developmental defects occur. Furthermore, zebrafish are ideally suited for the study of how vertebrate kidneys respond to injury and have provided researchers with invaluable information on repair processes after kidney injury. Importantly, zebrafish have profound potential for discovering treatment modalities and, in fact, studies in zebrafish models have provided leads for therapeutics for human patients suffering from kidney disease and kidney injury. Here, we discuss the similarities and differences in zebrafish and mammalian kidney models, and highlight some of the major contributions the zebrafish has made in the understanding of kidney development and disease. © 2017 Elsevier Inc. All rights reserved.

  13. Pleistophora hyphessobryconis (Microsporidia) infecting zebrafish (Danio rerio) in research facilities

    PubMed Central

    Sanders, Justin L; Lawrence, Christian; Nichols, Donald K; Brubaker, Jeffrey F.; Peterson, Tracy S; Murray, Katrina N.; Kent, Michael L

    2014-01-01

    Zebrafish (Danio rerio) are important models for biomedical research, and thus there is an increased concern about diseases afflicting them. Here we describe infections by Pleistophora hyphessobryconis (Microsporidia) in zebrafish from three laboratories. As reported in other aquarium fishes, affected zebrafish exhibited massive infections in the skeletal muscle, with no involvement of smooth or cardiac muscle. In addition, numerous spores within macrophages were observed in the visceral organs, including the ovaries. Transmission studies and ribosomal RNA (rRNA) gene sequence comparisons confirmed that the parasite from zebrafish was P. hyphessobryconis as described from neon tetra Paracheirodon innesi. Ten 15-day-old zebrafish were exposed to P. hyphessobryconis collected from one infected neon tetra, and 7 of 10 fish became infected. Comparison of P. hyphessobryconis small subunit rRNA gene sequence from neon tetra with that obtained from zebrafish was nearly identical, with < 1% difference. Given the severity of infections, P. hyphessobryconis should be added to the list of pathogens that should be avoided in zebrafish research facilities, and it would be prudent to not mix zebrafish used in research with other aquarium fishes. PMID:20853741

  14. Pleistophora hyphessobryconis (Microsporidia) infecting zebrafish Danio rerio in research facilities.

    PubMed

    Sanders, Justin L; Lawrence, Christian; Nichols, Donald K; Brubaker, Jeffrey F; Peterson, Tracy S; Murray, Katrina N; Kent, Michael L

    2010-07-26

    Zebrafish Danio rerio are important models for biomedical research, and thus, there is an increased concern about diseases afflicting them. Here we describe infections by Pleistophora hyphessobryconis (Microsporidia) in zebrafish from 3 laboratories. As reported in other aquarium fishes, affected zebrafish exhibited massive infections in the skeletal muscle, with no involvement of smooth or cardiac muscle. In addition, numerous spores within macrophages were observed in the visceral organs, including the ovaries. Transmission studies and ribosomal RNA (rRNA) gene sequence comparisons confirmed that the parasite from zebrafish was P. hyphessobryconis as described from neon tetra Paracheirodon innesi. Ten 15 d old zebrafish were exposed to P. hyphessobryconis collected from 1 infected neon tetra, and 7 of 10 fish became infected. Comparison of P. hyphessobryconis small subunit rRNA gene sequence from neon tetra with that obtained from zebrafish was nearly identical, with < 1% difference. Given the severity of infections, P. hyphessobryconis should be added to the list of pathogens that should be avoided in zebrafish research facilities, and it would be prudent to avoid mixing zebrafish used in research with other aquarium fishes.

  15. Zebrafish heart as a model for human cardiac electrophysiology

    PubMed Central

    Vornanen, Matti; Hassinen, Minna

    2016-01-01

    ABSTRACT The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart. PMID:26671745

  16. DRUG EFFECTS ON THE LOCOMOTOR ACTIVITY OF LARVAL ZEBRAFISH.

    EPA Science Inventory

    As part of an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae and the effects of prototype drugs. Zebrafish larvae (6-7 days post-fertilization) were indiv...

  17. Identifying Structural Alerts Based on Zebrafish Developmental Morphological Toxicity (TDS)

    EPA Science Inventory

    Zebrafish constitute a powerful alternative animal model for chemical hazard evaluation. To provide an in vivo complement to high-throughput screening data from the ToxCast program, zebrafish developmental toxicity screens were conducted on the ToxCast Phase I (Padilla et al., 20...

  18. Host-Pathogen Interactions Made Transparent with the Zebrafish Model

    PubMed Central

    Meijer, Annemarie H; Spaink, Herman P

    2011-01-01

    The zebrafish holds much promise as a high-throughput drug screening model for immune-related diseases, including inflammatory and infectious diseases and cancer. This is due to the excellent possibilities for in vivo imaging in combination with advanced tools for genomic and large scale mutant analysis. The context of the embryo’s developing immune system makes it possible to study the contribution of different immune cell types to disease progression. Furthermore, due to the temporal separation of innate immunity from adaptive responses, zebrafish embryos and larvae are particularly useful for dissecting the innate host factors involved in pathology. Recent studies have underscored the remarkable similarity of the zebrafish and human immune systems, which is important for biomedical applications. This review is focused on the use of zebrafish as a model for infectious diseases, with emphasis on bacterial pathogens. Following a brief overview of the zebrafish immune system and the tools and methods used to study host-pathogen interactions in zebrafish, we discuss the current knowledge on receptors and downstream signaling components that are involved in the zebrafish embryo’s innate immune response. We summarize recent insights gained from the use of bacterial infection models, particularly the Mycobacterium marinum model, that illustrate the potential of the zebrafish model for high-throughput antimicrobial drug screening. PMID:21366518

  19. FishNet: an online database of zebrafish anatomy.

    PubMed

    Bryson-Richardson, Robert J; Berger, Silke; Schilling, Thomas F; Hall, Thomas E; Cole, Nicholas J; Gibson, Abigail J; Sharpe, James; Currie, Peter D

    2007-08-17

    Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish. To overcome this deficit we have utilized the technique of optical projection tomography to produce three-dimensional (3D) models of larval fish. In order to view and display these models we have created FishNet http://www.fishnet.org.au, an interactive reference of zebrafish anatomy spanning the range of zebrafish development from 24 h until adulthood. FishNet contains more than 36,000 images of larval zebrafish, with more than 1,500 of these being annotated. The 3D models can be manipulated on screen or virtually sectioned. This resource represents the first complete embryo to adult atlas for any species in 3D.

  20. Direct visualization of replication dynamics in early zebrafish embryos.

    PubMed

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Okochi, Nanami; Hattori, Kaede; Ogata, Shin; Takebayashi, Shin-Ichiro; Ogata, Masato; Tamaru, Yutaka; Okumura, Katsuzumi

    2016-05-01

    We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.

  1. DRUG EFFECTS ON THE LOCOMOTOR ACTIVITY OF LARVAL ZEBRAFISH.

    EPA Science Inventory

    As part of an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae and the effects of prototype drugs. Zebrafish larvae (6-7 days post-fertilization) were indiv...

  2. Analysis of myostatin gene structure, expression and function in zebrafish.

    PubMed

    Xu, Cheng; Wu, Gang; Zohar, Yonathan; Du, Shao-Jun

    2003-11-01

    Myostatin is a member of the TGF-beta family that functions as a negative regulator of skeletal muscle development and growth in mammals. Recently, Myostatin has also been identified in fish; however, its role in fish muscle development and growth remains unknown. We have reported here the isolation and characterization of myostatin genomic gene from zebrafish and analysis of its expression in zebrafish embryos, larvae and adult skeletal muscles. Our data showed that myostatin was weakly expressed in early stage zebrafish embryos, and strongly expressed in swimming larvae, juvenile and skeletal muscles of adult zebrafish. Transient expression analysis revealed that the 1.2 kb zebrafish myostatin 5' flanking sequence could direct green fluorescent protein (GFP) expression predominantly in muscle cells, suggesting that the myostatin 5' flanking sequence contained regulatory elements required for muscle expression. To determine the biological function of Myostatin in fish, we generated a transgenic line that overexpresses the Myostatin prodomain in zebrafish skeletal muscles using a muscle-specific promoter. The Myostatin prodomain could act as a dominant negative and inhibit Myostatin function in skeletal muscles. Transgenic zebrafish expressing the Myostatin prodomain exhibited no significant change in myogenic gene expression and differentiation of slow and fast muscle cells at their embryonic stage. The transgenic fish, however, exhibited an increased number of myofibers in skeletal muscles, but no significant difference in fiber size. Together, these data demonstrate that Myostatin plays an inhibitory role in hyperplastic muscle growth in zebrafish.

  3. Transgenic zebrafish as sentinels for aquatic pollution.

    PubMed

    Carvan, M J; Dalton, T P; Stuart, G W; Nebert, D W

    2000-01-01

    Using the golden mutant zebrafish having a decrease in interfering pigmentation, we are developing transgenic lines in which DNA motifs that respond to selected environmental pollutants are capable of activating a reporter gene that can be easily assayed. We have begun with three response elements that recognize three important classes of foreign chemicals. Aromatic hydrocarbon response elements (AHREs) respond to numerous polycyclic hydrocarbons and halogenated coplanar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. Electrophile response elements (EPREs) respond to quinones and numerous other potent electrophilic oxidants. Metal response elements (MREs) respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. Soon, we will include estrogen response elements (EREs) to detect the effects of environmental endocrine disruptors, and retinoic acid response elements (RARE, RXRE) to detect the effects of retinoids in the environment. Each of these substances is known to be bioconcentrated in fish to varying degrees; for example, 10(-17) M TCDD in a body of water becomes concentrated to approximately 10(-12) M TCDD in a fish, where it would act upon the AHRE motif and turn on the luciferase (LUC) reporter gene. The living fish as a sentinel will not only be assayed intact in the luminometer, but--upon several days or weeks of depuration--would be usable again. To date, we have established that zebrafish transcription factors are able to recognize both mammalian and trout AHRE, EPRE, and MRE sequences in a dose-dependent and chemical-class-specific manner, and that expression of both the LUC and jellyfish green fluorescent protein (GFP) reporter genes is easily detected in zebrafish cell cultures and in the intact live zebrafish. Variations in sensitivity of this model system can be achieved by increasing the copy number of response elements and perhaps by altering the sequence of each core

  4. Scale development in zebrafish (Danio rerio)

    PubMed Central

    SIRE, JEAN-YVES; ALLIZARD, FRANCOISE; BABIAR, OLIVIER; BOURGUIGNON, JACQUELINE; QUILHAC, ALEXANDRA

    1997-01-01

    In the course of an extensive comparative, structural and developmental study of the cranial and postcranial dermal skeleton (teeth and scales) in osteichthyan fishes, we have undertaken investigations on scale development in zebrafish (Danio (Brachydanio) rerio) using alizarin red staining, and light and transmission electron microscopy. The main goal was to know whether zebrafish scales can be used as a model for further research on the processes controlling the development of the dermal skeleton in general, especially epithelial–mesenchymal interactions. Growth series of laboratory bred specimens were used to study in detail: (1) the relationship of scale appearance with size and age; (2) the squamation pattern; and (3) the events taking place in the epidermis and in the dermis, before and during scale initiation and formation, with the aim of searching for morphological indications of epithelial-mesenchymal interactions. Scales form late in ontogeny, generally when zebrafish are more than 8.0 mm in standard length. Within a population of zebrafish of the same age scale appearance is related to standard length, but when comparing populations of different age the size of the fish at scale appearance is also related to age. Scales always appear first in the posterior region of the body and the squamation then extends anteriorly. Scales develop in the dermis but closely apposed to the epidermal–dermal boundary. Cellular modifications occurring in the basal layer of the epidermis and in the dermis before scale formation clearly indicate that the basal epidermal cells differentiate first, before any evidence of differentiation of the progenitors of the scale-forming cells in the dermis. This strongly suggests that scale differentiation could be initiated by the epidermal basal layer cells which probably produce a molecular signal towards the dermis below. Subsequently dermal cells accumulate close to the epidermis, and differentiate to form scale papillae. The

  5. Detection of Autofluorescent Mycobacterium Chelonae in Living Zebrafish

    PubMed Central

    Whipps, Christopher M.; Moss, Larry G.; Sisk, Dana M.; Murray, Katrina N.; Tobin, David M.

    2014-01-01

    Abstract Mycobacterium chelonae is widespread in aquatic environments and can cause mycobacteriosis with low virulence in zebrafish. The risk of infection in zebrafish is exacerbated in closed-recirculating aquatic systems where rapidly growing mycobacteria can live on biofilms, as well as in zebrafish tissues. We have discovered a method of identifying and visualizing M. chelonae infections in living zebrafish using endogenous autofluorescence. Infected larvae are easily identified and can be excluded from experimental results. Because infection may reduce fertility in zebrafish, the visualization of active infection in contaminated eggs of transparent casper females simplifies screening. Transparent fish are also particularly useful as sentinels that can be examined periodically for the presence of autofluorescence, which can then be tested directly for M. chelonae. PMID:24451037

  6. Studying the immune response to human viral infections using zebrafish.

    PubMed

    Goody, Michelle F; Sullivan, Con; Kim, Carol H

    2014-09-01

    Humans and viruses have a long co-evolutionary history. Viral illnesses have and will continue to shape human history: from smallpox, to influenza, to HIV, and beyond. Animal models of human viral illnesses are needed in order to generate safe and effective antiviral medicines, adjuvant therapies, and vaccines. These animal models must support the replication of human viruses, recapitulate aspects of human viral illnesses, and respond with conserved immune signaling cascades. The zebrafish is perhaps the simplest, most commonly used laboratory model organism in which innate and/or adaptive immunity can be studied. Herein, we will discuss the current zebrafish models of human viral illnesses and the insights they have provided. We will highlight advantages of early life stage zebrafish and the importance of innate immunity in human viral illnesses. We will also discuss viral characteristics to consider before infecting zebrafish with human viruses as well as predict other human viruses that may be able to infect zebrafish.

  7. Development of sensory systems in zebrafish (Danio rerio)

    NASA Technical Reports Server (NTRS)

    Moorman, S. J.

    2001-01-01

    Zebrafish possess all of the classic sensory modalities: taste, tactile, smell, balance, vision, and hearing. For each sensory system, this article provides a brief overview of the system in the adult zebrafish followed by a more detailed overview of the development of the system. By far the majority of studies performed in each of the sensory systems of the zebrafish have involved some aspect of molecular biology or genetics. Although molecular biology and genetics are not major foci of the paper, brief discussions of some of the mutant strains of zebrafish that have developmental defects in each specific sensory system are included. The development of the sensory systems is only a small sampling of the work being done using zebrafish and provides a mere glimpse of the potential of this model for the study of vertebrate development, physiology, and human disease.

  8. Zebrafish Models of Human Liver Development and Disease

    PubMed Central

    Wilkins, Benjamin J.; Pack, Michael

    2016-01-01

    The liver performs a large number of essential synthetic and regulatory functions that are acquired during fetal development and persist throughout life. Their disruption underlies a diverse group of heritable and acquired diseases that affect both pediatric and adult patients. Although experimental analyses used to study liver development and disease are typically performed in cell culture models or rodents, the zebrafish is increasingly used to complement discoveries made in these systems. Forward and reverse genetic analyses over the past two decades have shown that the molecular program for liver development is largely conserved between zebrafish and mammals, and that the zebrafish can be used to model heritable human liver disorders. Recent work has demonstrated that zebrafish can also be used to study the mechanistic basis of acquired liver diseases. Here, we provide a comprehensive summary of how the zebrafish has contributed to our understanding of human liver development and disease. PMID:23897685

  9. Detection of autofluorescent Mycobacterium chelonae in living zebrafish.

    PubMed

    Whipps, Christopher M; Moss, Larry G; Sisk, Dana M; Murray, Katrina N; Tobin, David M; Moss, Jennifer B

    2014-02-01

    Mycobacterium chelonae is widespread in aquatic environments and can cause mycobacteriosis with low virulence in zebrafish. The risk of infection in zebrafish is exacerbated in closed-recirculating aquatic systems where rapidly growing mycobacteria can live on biofilms, as well as in zebrafish tissues. We have discovered a method of identifying and visualizing M. chelonae infections in living zebrafish using endogenous autofluorescence. Infected larvae are easily identified and can be excluded from experimental results. Because infection may reduce fertility in zebrafish, the visualization of active infection in contaminated eggs of transparent casper females simplifies screening. Transparent fish are also particularly useful as sentinels that can be examined periodically for the presence of autofluorescence, which can then be tested directly for M. chelonae.

  10. Neutrophils in host defense: new insights from zebrafish

    PubMed Central

    Harvie, Elizabeth A.; Huttenlocher, Anna

    2015-01-01

    Neutrophils are highly motile phagocytic cells that play a critical role in the immune response to infection. Zebrafish (Danio rerio) are increasingly used to study neutrophil function and host-pathogen interactions. The generation of transgenic zebrafish lines with fluorescently labeled leukocytes has made it possible to visualize the neutrophil response to infection in real time by use of optically transparent zebrafish larvae. In addition, the genetic tractability of zebrafish has allowed for the generation of models of inherited neutrophil disorders. In this review, we discuss several zebrafish models of infectious disease, both in the context of immunocompetent, as well as neutrophil-deficient hosts and how these models have shed light on neutrophil behavior during infection. PMID:25717145

  11. Zebrafish models for translational neuroscience research: from tank to bedside

    PubMed Central

    Stewart, Adam Michael; Braubach, Oliver; Spitsbergen, Jan; Gerlai, Robert; Kalueff, Allan V.

    2014-01-01

    The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes. PMID:24726051

  12. Acute caffeine administration affects zebrafish response to a robotic stimulus.

    PubMed

    Ladu, Fabrizio; Mwaffo, Violet; Li, Jasmine; Macrì, Simone; Porfiri, Maurizio

    2015-08-01

    Zebrafish has been recently proposed as a valid animal model to investigate the fundamental mechanisms regulating emotional behavior and evaluate the modulatory effects exerted by psychoactive compounds. In this study, we propose a novel methodological framework based on robotics and information theory to investigate the behavioral response of zebrafish exposed to acute caffeine treatment. In a binary preference test, we studied the response of caffeine-treated zebrafish to a replica of a shoal of conspecifics moving in the tank. A purely data-driven information theoretic approach was used to infer the influence of the replica on zebrafish behavior as a function of caffeine concentration. Our results demonstrate that acute caffeine administration modulates both the average speed and the interaction with the replica. Specifically, zebrafish exposed to elevated doses of caffeine show reduced locomotion and increased sensitivity to the motion of the replica. The methodology developed in this study may complement traditional experimental paradigms developed in the field of behavioral pharmacology.

  13. The zebrafish as a novel tool for cardiovascular drug discovery.

    PubMed

    Rocke, John; Lees, Julie; Packham, Ian; Chico, Timothy

    2009-01-01

    The zebrafish is a well established model of vertebrate development, but has recently emerged as a powerful tool for cardiovascular research and in vivo cardiovascular drug discovery. The zebrafish embryo's low cost, small size and permeability to small molecules coupled with the ability to generate thousands of embryos per week, and improved automation of assays of cardiovascular development and performance allow drug screening for a number of cardiovascular effects. Such studies have already led to discovery of novel cardiovascular drugs with potentially clinically beneficial effects. In this review we summarise the advantages and disadvantages of the zebrafish for drug discovery using some patents, previous literature on zebrafish-based drug screening and assess where the zebrafish will fit into existing drug discovery programmes.

  14. Modeling anxiety using adult zebrafish: A conceptual review

    PubMed Central

    Stewart, Adam; Gaikwad, Siddharth; Kyzar, Evan; Green, Jeremy; Roth, Andrew; Kalueff, Allan V.

    2011-01-01

    Zebrafish (Danio rerio) are rapidly emerging as a useful animal model in neurobehavioral research. Mounting evidence shows the suitability of zebrafish to model various aspects of anxiety-related states. Here, we evaluate established and novel approaches to uncover the molecular substrates, genetic pathways and neural circuits of anxiety using adult zebrafish. Experimental approaches to modeling anxiety in zebrafish include novelty-based paradigms, pharmacological and genetic manipulations, as well as innovative video-tracking, 3D-reconstructions and bioinformatics-based searchable databases and omics-based tools. Complementing traditional rodent models of anxiety, we provide a conceptual framework for the wider application of zebrafish and other aquatic models in anxiety research. PMID:21843537

  15. Host-microbe interactions in the developing zebrafish

    PubMed Central

    Kanther, Michelle; Rawls, John F.

    2010-01-01

    Summary of recent advances The amenability of the zebrafish to in vivo imaging and genetic analysis has fueled expanded use of this vertebrate model to investigate the molecular and cellular foundations of host-microbe relationships. Study of microbial encounters in zebrafish hosts has concentrated on developing embryonic and larval stages, when the advantages of the zebrafish model are maximized. A comprehensive understanding of these host-microbe interactions requires appreciation of the developmental context into which a microbe is introduced, as well as the effects of that microbial challenge on host ontogeny. In this review, we discuss how in vivo imaging and genetic analysis in zebrafish has advanced our knowledge of host-microbe interactions in the context of a developing vertebrate host. We focus on recent insights into immune cell ontogeny and function, commensal microbial relationships in the intestine, and microbial pathogenesis in zebrafish hosts. PMID:20153622

  16. [Potential of the zebrafish model to study congenital muscular dystrophies].

    PubMed

    Ryckebüsch, Lucile

    2015-10-01

    In order to better understand the complexity of congenital muscular dystrophies (CMD) and develop new strategies to cure them, it is important to establish new disease models. Due to its numerous helpful attributes, the zebrafish has recently become a very powerful animal model for the study of CMD. For some CMD, this vertebrate model is phenotypically closer to human pathology than the murine model. Over the last few years, researchers have developed innovative techniques to screen rapidly and on a large scale for muscle defects in zebrafish. Furthermore, new genome editing techniques in zebrafish make possible the identification of new disease models. In this review, the major attributes of zebrafish for CMD studies are discussed and the principal models of CMD in zebrafish are highlighted.

  17. Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology

    PubMed Central

    Challa, Anil Kumar

    2013-01-01

    Abstract Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  18. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

  19. UNUSUAL FINDINGS IN ZEBRAFISH, DANIO RERIO, FROM TOXICOLOGICAL STUDIES AND THE ZEBRAFISH INTERNATIONAL RESOURCE CENTER DIAGNOSTIC SERVICE

    EPA Science Inventory

    A number of interesting and unusual lesions have been diagnosed in zebrafish that have been evaluated from toxicological studies or submitted as cases to the Diagnostic Service at Oregon State University. Lesions were observed in various wild-type and mutant lines of zebrafish an...

  20. Stimulus dependence of the development of the zebrafish (Danio rerio) vestibular system.

    PubMed

    Moorman, S J; Burress, C; Cordova, R; Slater, J

    1999-02-05

    It has been suggested that stimulus dependence is a general feature of all developing sensory systems. We tested this idea for the developing zebrafish vestibular system using a bioreactor the National Aeronautic and Space Agency designed to simulate microgravity for cells in culture on earth. We replaced the culture medium with aquarium water and maintained zebrafish eggs/hatchlings in the bioreactor for either 72 or 96 h postfertilization. These experimental animals displayed a swimming behavior that was indistinguishable from the control animals when illuminated from above. However, when illuminated from below, experimental animals swam not only dorsal surface up, but also lying on their side; they corkscrewed, swam vertical loops, and occasionally even swam upside down. When incubated in the bioreactor for 96 h, the saccular otolith was significantly smaller than normal, suggesting that otolith development was either delayed or slower than normal. When incubated in the bioreactor for 72 h, some animals were missing one or more otoliths. In contrast, control animals all had two otoliths on each side. This supports the idea that otolith development was delayed. Immediately upon removal from the bioreactor at 96 h, experimental animals showed some signs of compensatory eye rotation, but with a much less clear relationship between the orientation of the eye and the direction of gravity than the age-matched control animals. This difference was still obvious 1 day later. These results support the idea that development of the vestibular system in zebrafish is dependent on the presence of the normal stimulus the system is designed to detect.

  1. A Dominant Negative Zebrafish Ahr2 Partially Protects Developing Zebrafish from Dioxin Toxicity

    PubMed Central

    Lanham, Kevin A.; Prasch, Amy L.; Weina, Kasia M.; Peterson, Richard E.; Heideman, Warren

    2011-01-01

    The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is thought to be caused by activation of the aryl hydrocarbon receptor (AHR). However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs). This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity. PMID:22194803

  2. Embryotoxicity and biotransformation responses in zebrafish exposed to water-soluble fraction of crude oil.

    PubMed

    Pauka, Luciana M; Maceno, Marcell; Rossi, Stefani C; Silva de Assis, Helena C

    2011-04-01

    The toxic effects of water-soluble fraction (WSF) of crude oil (API27, Petrobras Campos Basin, Brazil) were evaluated during the early life stages of zebrafish, as well as its biotransformation in juvenile fish. Embryonic development was studied during 96 h. Reduced heartbeat rate, weak pigmentation, tail defects, and embryo mortality were observed for all of the tested concentrations of the WSF. Activities of the biotransformation enzymes were induced at the highest concentrations, showing that these enzymes played a role in its elimination. As shown in this study the crude oil WSF altered the normal embryonic development of fish.

  3. Multilayer mounting for long-term light sheet microscopy of zebrafish.

    PubMed

    Weber, Michael; Mickoleit, Michaela; Huisken, Jan

    2014-02-27

    Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

  4. Expression of mt2 and smt-B upon cadmium exposure and cold shock in zebrafish (Danio rerio).

    PubMed

    Wu, Su Mei; Zheng, Yu Da; Kuo, Chien-Hsien

    2008-08-01

    Metallothionein-2 (mt2) and similar to metallothionein-B (smt-B) are included in the MT gene family. The objective of this study was to compare mt2 and smt-B messenger (m)RNA expressions after cadmium exposure and cold shock with whole-mount in situ hybridization in immature zebrafish (Danio rerio) and with a semi-quantitative RT-PCR in mature zebrafish. Three-day post-fertilization (dpf) larvae were treated with 0, 0.08, 0.26, and 0.89 microM cadmium for 24 and 48 h, and some larvae were challenged with a normal (28.5 degrees C) or low temperature (12 degrees C) for 12, 24, and 48 h. Results were obtained. (1) During embryonic and larval development, mt2 mRNA existed at 6 h post-fertilization (hpf), and the level rapidly increased to 24 hpf, then it gradually increased with further larval growth. smt-B was found at 12 hpf, and it also rapidly increased to 24 hpf, but remained constant during further larval development. (2) The mt2 mRNA signals and whole-body Cd contents displayed dose- and time-dependent responses after Cd exposure. After cold shock, mt2 mRNA signals also showed time-dependent expression. But smt-B mRNA signals were not appeared by either challenge. Besides, mature zebrafish were treated with 1.78 microM Cd and found that the highest levels of smt-B mRNA (smt-B/beta-actin) appeared in brain, and seems a reverse expression between smt-B mRNA and mt2 in brain after Cd exposure. Apparently, mt2 is possibly more relevant to Cd detoxification and cold shock adaptation in zebrafish larvae compared to smt-B, but smt-B might be related to certain physiological functions in neural (or brain) of mature zebrafish.

  5. Intrinsic regulation of sinoatrial node function and the zebrafish as a model of stretch effects on pacemaking.

    PubMed

    MacDonald, Eilidh A; Stoyek, Matthew R; Rose, Robert A; Quinn, T Alexander

    2017-07-22

    Excitation of the heart occurs in a specialised region known as the sinoatrial node (SAN). Tight regulation of SAN function is essential for the maintenance of normal heart rhythm and the response to (patho-)physiological changes. The SAN is regulated by extrinsic (central nervous system) and intrinsic (neurons, peptides, mechanics) factors. The positive chronotropic response to stretch in particular is essential for beat-by-beat adaptation to changes in hemodynamic load. Yet, the mechanism of this stretch response is unknown, due in part to the lack of an appropriate experimental model for targeted investigations. We have been investigating the zebrafish as a model for the study of intrinsic regulation of SAN function. In this paper, we first briefly review current knowledge of the principal components of extrinsic and intrinsic SAN regulation, derived primarily from experiments in mammals, followed by a description of the zebrafish as a novel experimental model for studies of intrinsic SAN regulation. This mini-review is followed by an original investigation of the response of the zebrafish isolated SAN to controlled stretch. Stretch causes an immediate and continuous increase in beating rate in the zebrafish isolated SAN. This increase reaches a maximum part way through a period of sustained stretch, with the total change dependent on the magnitude and direction of stretch. This is comparable to what occurs in isolated SAN from most mammals (including human), suggesting that the zebrafish is a novel experimental model for the study of mechanisms involved in the intrinsic regulation of SAN function by mechanical effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Early life stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish (Danio rerio)

    SciTech Connect

    Henry, T.R.; Hornung, M.W.; Abnet, C.C.; Peterson, R.E.

    1995-12-31

    TCDD and related compounds cause toxicity in fish early life stages, characterized by edema, regional ischemia, craniofacial malformations, growth retardation and mortality. Determining the mechanism of these effects requires understanding normal early life stage development, which has been studied extensively in the zebrafish. Establishing zebrafish as a model for TCDD developmental toxicity requires demonstration that TCDD adversely affects zebrafish early life stages. Toxicity of TCDD to zebrafish early life stages was characterized by exposing newly fertilized eggs for 1 hr to water containing acetone or graded concentrations of [{sup 3}H]-TCDD and observed for signs of toxicity at 12 hr intervals for 240 hr post fertilization (hpf). TCDD did not increase embryo mortality during the egg stage (0--48 hpf) nor did it affect the time to hatching (48--96 hpf). At the highest TCDD egg doses (4.5--6.5 ng/g) the earliest sign of toxicity was pericardial edema (72 hpf) followed by the onset of yolk sac edema (96 hpf) onset of mortality (132 hpf). At lower egg doses the same effects were seen but after a longer delay period. Other signs of toxicity included craniofacial malformations, cranial edema and loss of swimming activity prior to death. To determine the dose-response relationship for pericardial and yolk sac edema and larval mortality the cumulative incidence of each effect was determined at 240 hpf. The ED{sub 50}s (95% fiducial limits) for pericardial edema and yolk sac edema were 2.1 6 (1.82--2.48) and 2.43 (2.12--2.72) ng TCDD/g egg, respectively. The LD{sub 50} was 2.45 (1.94--2.89) ng TCDD/g egg. In conclusion, the signs of TCDD early life stage toxicity in zebrafish are essentially identical to those in other fish species, however, larger egg doses of TCDD are required to elicit the effects.

  7. 8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

    SciTech Connect

    Yan, Lifeng; Zhou, Yong; Yu, Shanhe; Ji, Guixiang; Liu, Wei; Gu, Aihua

    2013-11-15

    Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5{sup +} cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits. - Highlights: • A key DNA repair enzyme ogg1 is expressed in the embryonic heart in zebrafish. • We found that ogg1 is essential for normal cardiac morphogenesis in zebrafish. • The production of embryonic cardiomyocytes requires appropriate ogg1 expression. • Ogg1 critically regulated proliferation of cardiac progenitor cells in zebrafish. • foxh1 is a partner of ogg1 in the cardiac development in response to DNA damage.

  8. Acute and long-term effects after single loading of functionalized multi-walled carbon nanotubes into zebrafish (Danio rerio)

    SciTech Connect

    Cheng Jinping; Chan, C.M.; Veca, L. Monica; Poon, W.L.; Chan, P.K.; Qu Liangwei; Sun Yaping Cheng, S.H.

    2009-03-01

    Carbon nanotubes (CNTs) are widely explored for biomedical applications, but there is very limited information regarding their in vivo biodistribution and biocompatibility. Here, we report the in vivo biodistribution and long-term effects of functionalized multi-walled carbon nanotubes (MWCNTs) in developing zebrafish. The fluorescent-labeled MWCNTs were introduced into zebrafish embryos at 1-cell stage and at 72 h post fertilization through microinjection. After single injection, both acute and long-term interactions between zebrafish and functionalized MWCNTs were studied. The injected FITC-BSA-MWCNTs (at 1-cell stage) were allocated to all blastoderm cells of the embryos through proliferation, and were distinctively excluded from the yolk cell. When introduced into the circulation system, FITC-BSA-MWCNTs moved easily in the compartments and finally were cleaned out by the body at 96 h after the loading. At early stages, the treated zebrafish embryos generated immune response by accumulating circulating white blood cells at the trunk region. Under transmission electron microscope, many lysosome-like vesicles were observed in the blastoderm cells of the treated embryos. The zebrafish loaded with MWCNTs had normal primordial germ cells at early stage and produced second generation later on. However, the larvae of the second generation had obviously lower survival rates as compared to the untreated groups, suggesting a negative effect on the reproduction potential. These results suggest that extensive purification and functionalization processes can help improve the biocompatibility of CNTs. This study also indicates that purified CNTs may have long-term toxicity effects when they were delivered into the body.

  9. Lactobacillus rhamnosus Accelerates Zebrafish Backbone Calcification and Gonadal Differentiation through Effects on the GnRH and IGF Systems

    PubMed Central

    Avella, Matteo A.; Place, Allen; Du, Shao-Jun; Williams, Ernest; Silvi, Stefania; Zohar, Yonathan; Carnevali, Oliana

    2012-01-01

    Endogenous microbiota play essential roles in the host’s immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host’s development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application. PMID:23029107

  10. The Hippo Pathway Controls a Switch between Retinal Progenitor Cell Proliferation and Photoreceptor Cell Differentiation in Zebrafish

    PubMed Central

    Asaoka, Yoichi; Hata, Shoji; Namae, Misako; Furutani-Seiki, Makoto; Nishina, Hiroshi

    2014-01-01

    The precise regulation of numbers and types of neurons through control of cell cycle exit and terminal differentiation is an essential aspect of neurogenesis. The Hippo signaling pathway has recently been identified as playing a crucial role in promoting cell cycle exit and terminal differentiation in multiple types of stem cells, including in retinal progenitor cells. When Hippo signaling is activated, the core Mst1/2 kinases activate the Lats1/2 kinases, which in turn phosphorylate and inhibit the transcriptional cofactor Yap. During mouse retinogenesis, overexpression of Yap prolongs progenitor cell proliferation, whereas inhibition of Yap decreases this proliferation and promotes retinal cell differentiation. However, to date, it remains unknown how the Hippo pathway affects the differentiation of distinct neuronal cell types such as photoreceptor cells. In this study, we investigated whether Hippo signaling regulates retinogenesis during early zebrafish development. Knockdown of zebrafish mst2 induced early embryonic defects, including altered retinal pigmentation and morphogenesis. Similar abnormal retinal phenotypes were observed in zebrafish embryos injected with a constitutively active form of yap [(yap (5SA)]. Loss of Yap’s TEAD-binding domain, two WW domains, or transcription activation domain attenuated the retinal abnormalities induced by yap (5SA), indicating that all of these domains contribute to normal retinal development. Remarkably, yap (5SA)-expressing zebrafish embryos displayed decreased expression of transcription factors such as otx5 and crx, which orchestrate photoreceptor cell differentiation by activating the expression of rhodopsin and other photoreceptor cell genes. Co-immunoprecipitation experiments revealed that Rx1 is a novel interacting partner of Yap that regulates photoreceptor cell differentiation. Our results suggest that Yap suppresses the differentiation of photoreceptor cells from retinal progenitor cells by repressing Rx1

  11. Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

    PubMed

    Avella, Matteo A; Place, Allen; Du, Shao-Jun; Williams, Ernest; Silvi, Stefania; Zohar, Yonathan; Carnevali, Oliana

    2012-01-01

    Endogenous microbiota play essential roles in the host's immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host's development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment) with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf) exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP), higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group). We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

  12. Longitudinal visualization of vascular occlusion, reperfusion, and remodeling in a zebrafish model of retinal vascular leakage using OCT angiography

    NASA Astrophysics Data System (ADS)

    Spitz, Kathleen; Bozic, Ivan; Desai, Vineet; Rao, Gopikrishna M.; Pollock, Lana M.; Anand-Apte, Bela; Tao, Yuankai K.

    2017-02-01

    Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are two of the leading causes of blindness and visual impairment in the world. Neovascularization results in severe vision loss in DR and AMD and, thus, there is an unmet need to identify mechanisms of pathogenesis and novel anti-angiogenic therapies. Zebrafish is a leading model organism for studying human disease pathogenesis, and the highly conserved drug activity between zebrafish and humans and their ability to readily absorb small molecules dissolved in water has benefited pharmaceutical discovery. Here, we use optical coherence tomography (OCT) and OCT angiography (OCT-A) to perform noninvasive, in vivo retinal imaging in a zebrafish model of vascular leakage. Zebrafish were treated with diethylaminobenzaldehyde (DEAB) to induce vascular leakage and imaged with OCT and OCT-A at six time points over two weeks: baseline one day before treatment and one, three, six, eight, and ten days post treatment. Longitudinal functional imaging showed significant vascular response immediately after DEAB treatment. Observed vascular changes included partial or complete vascular occlusion immediately after treatment and reperfusion during a two-week period. Increased vascular tortuosity several days post treatment indicated remodeling, and bifurcations and collateral vessel formation were also observed. In addition, significant treatment response variabilities were observed in the contralateral eye of the same animal. Anatomical and functional normalization was observed in most animals by ten days post treatment. These preliminary results motivate potential applications of OCT-A as a tool for studying pathogenesis and therapeutic screening in zebrafish models of retinal vascular disease.

  13. Zebrafish: an animal model for research in veterinary medicine.

    PubMed

    Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

    2015-01-01

    The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic

  14. Generation of Alzheimer's Disease Transgenic Zebrafish Expressing Human APP Mutation Under Control of Zebrafish appb Promotor.

    PubMed

    Pu, Yun-Zhu; Liang, Liang; Fu, Ai-Ling; Liu, Yan; Sun, Lan; Li, Qian; Wu, Dan; Sun, Man-Ji; Zhang, Ying-Ge; Zhao, Bao-Quan

    2017-01-01

    Amyloid peptide precursor (APP) as the precursor protein of peptide betaamyloid (β-amyloid, Aβ), which is thought to play a central role in the pathogenesis of Alzheimer's disease (AD), also has an important effect on the development and progression of AD. Through knocking-in APP gene in animals, numerous transgenic AD models have been set up for the investigation of the mechanisms behind AD pathogenesis and the screening of anti-AD drugs. However, there are some limitations to these models and here is a need for such an AD model that is economic as well as has satisfactory genetic homology with human. We generated a new AD transgenic model by knocking a mutant human APP gene (APPsw) in zebrafish with appb promoter of zebrafish to drive the expression of APPsw. Fluorescent image and immunochemistry stain showed and RT-PCR and western blot assay confirmed that APPsw was successfully expressed in the brain, heart, eyes and vasculature of the transgenic zebrafish. Behavioral observation demonstrated that the transgenic zebrafish had AD-like symptoms. Histopathological observation found that there were cerebral β-amyloidosis and angiopathy (CAA), which induced neuron loss and enlarged pervascular space. These results suggest that APPsw transgenic zebrafish well simulate the pathological characters of AD and can be used as an economic AD transgenic model. Furthermore, the new model suggested that APP can express in microvasculatures and cause the Aβ generation and deposition in cerebral vessel which further destroys cerebral vascular structure resulting in the development and/or the progress of AD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Multivariate normality

    NASA Technical Reports Server (NTRS)

    Crutcher, H. L.; Falls, L. W.

    1976-01-01

    Sets of experimentally determined or routinely observed data provide information about the past, present and, hopefully, future sets of similarly produced data. An infinite set of statistical models exists which may be used to describe the data sets. The normal distribution is one model. If it serves at all, it serves well. If a data set, or a transformation of the set, representative of a larger population can be described by the normal distribution, then valid statistical inferences can be drawn. There are several tests which may be applied to a data set to determine whether the univariate normal model adequately describes the set. The chi-square test based on Pearson's work in the late nineteenth and early twentieth centuries is often used. Like all tests, it has some weaknesses which are discussed in elementary texts. Extension of the chi-square test to the multivariate normal model is provided. Tables and graphs permit easier application of the test in the higher dimensions. Several examples, using recorded data, illustrate the procedures. Tests of maximum absolute differences, mean sum of squares of residuals, runs and changes of sign are included in these tests. Dimensions one through five with selected sample sizes 11 to 101 are used to illustrate the statistical tests developed.

  16. N-cadherin is essential for retinal lamination in the zebrafish.

    PubMed

    Erdmann, Bettina; Kirsch, Frank-P; Rathjen, Fritz G; Moré, Margret I

    2003-03-01

    N-cadherin is one of the major Ca(2+)-dependent cell adhesion proteins in the developing nervous system. Here, we analyze eye development in the zebrafish N-cadherin loss-of-function mutant parachute(paR2.10) (pac(paR2.10)). The zebrafish visual system is fully developed by the time pac(paR2.10) mutants show lethality at day 5. Already at 24 hr postfertilization (hpf), mutant retinal cells are more disorganized and more rounded than in wild-type. At later stages, mutant retinae display a severe lamination defect with rosette formation (mostly islands of plexiform layer tissue surrounded by inner nuclear layer or photoreceptor cells), even though all major classes of cell types appear to be present as determined by histology. Of interest, electron microscopy reveals that the islands of plexiform layer tissue contain a normal amount of synapses with normal morphology. Although mutant photoreceptor cells are sometimes deformed, all typical structural components are present, including the membranous discs for rhodopsin storage. The lens fibers of the pac(paR2.10) mutants develop completely normally, but in some cases, lens epithelial cells round up and become multilayered. We conclude that cell adhesion mediated by N-cadherin is of major importance for retinal lamination and involved in maintenance of the lens epithelial sheet, but is not essential for the formation of photoreceptor ultrastructure or for synaptogenesis.

  17. Novel biomarkers of perchlorate exposure in zebrafish

    USGS Publications Warehouse

    Mukhi, S.; Carr, J.A.; Anderson, T.A.; Patino, R.

    2005-01-01

    Perchlorate inhibits iodide uptake by thyroid follicles and lowers thyroid hormone production. Although several effects of perchlorate on the thyroid system have been reported, the utility of these pathologies as markers of environmental perchlorate exposures has not been adequately assessed. The present study examined time-course and concentration-dependent effects of perchlorate on thyroid follicle hypertrophy, colloid depletion, and angiogenesis; alterations in whole-body thyroxine (T4) levels; and somatic growth and condition factor of subadult and adult zebrafish. Changes in the intensity of the colloidal T4 ring previously observed in zebrafish also were examined immunohistochemically. Three-month-old zebrafish were exposed to ammonium perchlorate at measured perchlorate concentrations of 0, 11, 90, 1,131, and 11,480 ppb for 12 weeks and allowed to recover in clean water for 12 weeks. At two weeks of exposure, the lowest-observed-effective concentrations (LOECs) of perchlorate that induced angiogenesis and depressed the intensity of colloidal T4 ring were 90 and 1,131 ppb, respectively; other parameters were not affected (whole-body T4 was not determined at this time). At 12 weeks of exposure, LOECs for colloid depletion, hypertrophy, angiogenesis, and colloidal T4 ring were 11,480, 1,131, 90, and 11 ppb, respectively. All changes were reversible, but residual effects on angiogenesis and colloidal T4 ring intensity were still present after 12 weeks of recovery (LOEC, 11,480 ppb). Whole-body T 4 concentration, body growth (length and weight), and condition factor were not affected by perchlorate. The sensitivity and longevity of changes in colloidal T4 ring intensity and angiogenesis suggest their usefulness as novel markers of perchlorate exposure. The 12-week LOEC for colloidal T4 ring is the lowest reported for any perchlorate biomarker in aquatic vertebrates. ?? 2005 SETAC.

  18. Learning and memory in zebrafish (Danio rerio).

    PubMed

    Gerlai, R

    2016-01-01

    Learning and memory are defining features of our own species inherently important to our daily lives and to who we are. Without our memories we cease to exist as a person. Without our ability to learn individuals and collectively our society would cease to function. Diseases of the mind still remain incurable. The interest in understanding of the mechanisms of learning and memory is thus well founded. Given the complexity of such mechanisms, concerted efforts have been made to study them under controlled laboratory conditions, ie, with laboratory model organisms. The zebrafish, although new in this field, is one such model organism. The rapidly developing forward- and reverse genetic methods designed for the zebrafish and the increasing use of pharmacological tools along with numerous neurobiology techniques make this species perhaps the best model for the analysis of the mechanisms of complex central nervous system characteristics. The fact that it is an evolutionarily ancient and simpler vertebrate, but at the same time it possesses numerous conserved features across multiple levels of biological organization makes this species an excellent tool for the analysis of the mechanisms of learning and memory. The bottleneck lies in our understanding of its cognitive and mnemonic features, the topic of this chapter. The current paper builds on a chapter published in the previous edition and continues to focus on associative learning, but now it extends the discussion to other forms of learning and to recent discoveries on memory-related features and findings obtained both in adults and larval zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Toxicity of chlorine to zebrafish embryos

    PubMed Central

    Kent, Michael L.; Buchner, Cari; Barton, Carrie; Tanguay, Robert L.

    2014-01-01

    Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish (Danio rerio) research laboratories. Most laboratories use approximately 25 – 50 ppm unbuffered chlorine solution for 5 – 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH. Consistent with this, we found that unbufferred chlorine solutions (pH ca 8–9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure. PMID:24429474

  20. Adaptive locomotor behavior in larval zebrafish.

    PubMed

    Portugues, Ruben; Engert, Florian

    2011-01-01

    In this study we report that larval zebrafish display adaptive locomotor output that can be driven by unexpected visual feedback. We develop a new assay that addresses visuomotor integration in restrained larval zebrafish. The assay involves a closed-loop environment in which the visual feedback a larva receives depends on its own motor output in a way that resembles freely swimming conditions. The experimenter can control the gain of this closed feedback loop, so that following a given motor output the larva experiences more or less visual feedback depending on whether the gain is high or low. We show that increases and decreases in this gain setting result in adaptive changes in behavior that lead to a generalized decrease or increase of motor output, respectively. Our behavioral analysis shows that both the duration and tail beat frequency of individual swim bouts can be modified, as well as the frequency with which bouts are elicited. These changes can be implemented rapidly, following an exposure to a new gain of just 175 ms. In addition, modifications in some behavioral parameters accumulate over tens of seconds and effects last for at least 30 s from trial to trial. These results suggest that larvae establish an internal representation of the visual feedback expected from a given motor output and that the behavioral modifications are driven by an error signal that arises from the discrepancy between this expectation and the actual visual feedback. The assay we develop presents a unique possibility for studying visuomotor integration using imaging techniques available in the larval zebrafish.

  1. Acclimation of zebrafish to transport stress.

    PubMed

    Dhanasiri, Anusha K S; Fernandes, Jorge M O; Kiron, Viswanath

    2013-03-01

    Welfare of fish is commonly neglected when they are transported. This study examines the effect of a 72-h mock transport on certain aspects of the stress physiology of two groups of zebrafish-the first transported in water enriched with a nitrifying bacterial consortium and the second in water without the enrichment. Zebrafish were examined at different time points-before packing (BP), immediately after packing them in transport bags (AP), at the end of transport (AT), and 72 h thereafter (PT)-to assess the primary (cortisol) and secondary (glucose) stress responses. In addition, the relevant genes in hypothalamic-pituitary-interrenal (HPI) axis (crf in brain, mc2r, star, cyp11c1, and hsd11b2 in kidney), including that of mineralocorticoid receptor (mr in kidney), were studied. Procedures during packing caused an increase in whole body cortisol levels of both fish groups. Only in the fish transported without the bacterial consortium, an increase in the levels of whole body cortisol as well as blood glucose was observed at the end of the transport. At the same time point and in the same fish group, the transcripts of mr and hsd11b2 were enhanced, probably to cope with the stress and to maintain homeostasis. The mRNA levels of the other genes in the HPI stress axis (crf, mc2r, star, and cyp11c1) were not significantly altered. Zebrafish transported in water enriched with the bacterial consortium exhibited a speedier stress acclimation. Nevertheless, only through in-depth studies the beneficial effect of the consortium can be confirmed.

  2. montalcino, a Zebrafish Model for Variegate Porphyria

    PubMed Central

    Dooley, Kimberly A.; Fraenkel, Paula G.; Langer, Nathaniel B.; Schmid, Bettina; Davidson, Alan J.; Weber, Gerhard; Chiang, Ken; Foott, Helen; Dwyer, Caitlin; Wingert, Rebecca A.; Zhou, Yi; Paw, Barry H.; Zon, Leonard I.

    2008-01-01

    Objective Inherited or acquired mutations in the heme biosynthetic pathway lead to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant. Methods Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with RT-PCR was utilized to identify the genetic mutation, which was confirmed via allele specific oligo hybridizations. Whole mount in situ hybridizations and 0-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype. Results Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hpf are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox. Conclusion In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria. PMID:18550261

  3. Premature aging in telomerase-deficient zebrafish

    PubMed Central

    Anchelin, Monique; Alcaraz-Pérez, Francisca; Martínez, Carlos M.; Bernabé-García, Manuel; Mulero, Victoriano; Cayuela, María L.

    2013-01-01

    SUMMARY The study of telomere biology is crucial to the understanding of aging and cancer. In the pursuit of greater knowledge in the field of human telomere biology, the mouse has been used extensively as a model. However, there are fundamental differences between mouse and human cells. Therefore, additional models are required. In light of this, we have characterized telomerase-deficient zebrafish (Danio rerio) as the second vertebrate model for human telomerase-driven diseases. We found that telomerase-deficient zebrafish show p53-dependent premature aging and reduced lifespan in the first generation, as occurs in humans but not in mice, probably reflecting the similar telomere length in fish and humans. Among these aging symptoms, spinal curvature, liver and retina degeneration, and infertility were the most remarkable. Although the second-generation embryos died in early developmental stages, restoration of telomerase activity rescued telomere length and survival, indicating that telomerase dosage is crucial. Importantly, this model also reproduces the disease anticipation observed in humans with dyskeratosis congenita (DC). Thus, telomerase haploinsufficiency leads to anticipation phenomenon in longevity, which is related to telomere shortening and, specifically, with the proportion of short telomeres. Furthermore, p53 was induced by telomere attrition, leading to growth arrest and apoptosis. Importantly, genetic inhibition of p53 rescued the adverse effects of telomere loss, indicating that the molecular mechanisms induced by telomere shortening are conserved from fish to mammals. The partial rescue of telomere length and longevity by restoration of telomerase activity, together with the feasibility of the zebrafish for high-throughput chemical screening, both point to the usefulness of this model for the discovery of new drugs able to reactivate telomerase in individuals with DC. PMID:23744274

  4. Adaptive Locomotor Behavior in Larval Zebrafish

    PubMed Central

    Portugues, Ruben; Engert, Florian

    2011-01-01

    In this study we report that larval zebrafish display adaptive locomotor output that can be driven by unexpected visual feedback. We develop a new assay that addresses visuomotor integration in restrained larval zebrafish. The assay involves a closed-loop environment in which the visual feedback a larva receives depends on its own motor output in a way that resembles freely swimming conditions. The experimenter can control the gain of this closed feedback loop, so that following a given motor output the larva experiences more or less visual feedback depending on whether the gain is high or low. We show that increases and decreases in this gain setting result in adaptive changes in behavior that lead to a generalized decrease or increase of motor output, respectively. Our behavioral analysis shows that both the duration and tail beat frequency of individual swim bouts can be modified, as well as the frequency with which bouts are elicited. These changes can be implemented rapidly, following an exposure to a new gain of just 175 ms. In addition, modifications in some behavioral parameters accumulate over tens of seconds and effects last for at least 30 s from trial to trial. These results suggest that larvae establish an internal representation of the visual feedback expected from a given motor output and that the behavioral modifications are driven by an error signal that arises from the discrepancy between this expectation and the actual visual feedback. The assay we develop presents a unique possibility for studying visuomotor integration using imaging techniques available in the larval zebrafish. PMID:21909325

  5. Proteomic analysis of zebrafish caudal fin regeneration.

    PubMed

    Saxena, Sandeep; Singh, Sachin K; Lakshmi, Mula G Meena; Meghah, Vuppalapaty; Bhatti, Bhawna; Swamy, Cherukuvada V Brahmendra; Sundaram, Curam S; Idris, Mohammed M

    2012-06-01

    The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal α1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism.

  6. Zebrafish: A Versatile Animal Model for Fertility Research.

    PubMed

    Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun; Goh, Bey Hing

    2016-01-01

    The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.

  7. The zebrafish world of colors and shapes: preference and discrimination.

    PubMed

    Oliveira, Jessica; Silveira, Mayara; Chacon, Diana; Luchiari, Ana

    2015-04-01

    Natural environment imposes many challenges to animals, which have to use cognitive abilities to cope with and exploit it to enhance their fitness. Since zebrafish is a well-established model for cognitive studies and high-throughput screening for drugs and diseases that affect cognition, we tested their ability for ambient color preference and 3D objects discrimination to establish a protocol for memory evaluation. For the color preference test, zebrafish were observed in a multiple-chamber tank with different environmental color options. Zebrafish showed preference for blue and green, and avoided yellow and red. For the 3D objects discrimination, zebrafish were allowed to explore two equal objects and then observed in a one-trial test in which a new color, size, or shape of the object was presented. Zebrafish showed discrimination for color, shape, and color+shape combined, but not size. These results imply that zebrafish seem to use some categorical system to discriminate items, and distracters affect their ability for discrimination. The type of variables available (color and shape) may favor zebrafish objects perception and facilitate discrimination processing. We suggest that this easy and simple memory test could serve as a useful screening tool for cognitive dysfunction and neurotoxicological studies.

  8. A sequence-based variation map of zebrafish.

    PubMed

    Patowary, Ashok; Purkanti, Ramya; Singh, Meghna; Chauhan, Rajendra; Singh, Angom Ramcharan; Swarnkar, Mohit; Singh, Naresh; Pandey, Vikas; Torroja, Carlos; Clark, Matthew D; Kocher, Jean-Pierre; Clark, Karl J; Stemple, Derek L; Klee, Eric W; Ekker, Stephen C; Scaria, Vinod; Sivasubbu, Sridhar

    2013-03-01

    Zebrafish (Danio rerio) is a popular vertebrate model organism largely deployed using outbred laboratory animals. The nonisogenic nature of the zebrafish as a model system offers the opportunity to understand natural variations and their effect in modulating phenotype. In an effort to better characterize the range of natural variation in this model system and to complement the zebrafish reference genome project, the whole genome sequence of a wild zebrafish at 39-fold genome coverage was determined. Comparative analysis with the zebrafish reference genome revealed approximately 5.2 million single nucleotide variations and over 1.6 million insertion-deletion variations. This dataset thus represents a new catalog of genetic variations in the zebrafish genome. Further analysis revealed selective enrichment for variations in genes involved in immune function and response to the environment, suggesting genome-level adaptations to environmental niches. We also show that human disease gene orthologs in the sequenced wild zebrafish genome show a lower ratio of nonsynonymous to synonymous single nucleotide variations.

  9. Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

    PubMed Central

    Cachat, Jonathan; Stewart, Adam; Utterback, Eli; Hart, Peter; Gaikwad, Siddharth; Wong, Keith; Kyzar, Evan; Wu, Nadine; Kalueff, Allan V.

    2011-01-01

    The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior. PMID:21408171

  10. Functionally conserved effects of rapamycin exposure on zebrafish.

    PubMed

    Sucularli, Ceren; Shehwana, Huma; Kuscu, Cem; Dungul, Dilay Ciglidag; Ozdag, Hilal; Konu, Ozlen

    2016-05-01

    Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a well‑known anti‑cancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Meta‑analysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dose‑dependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycin‑modulated functional pathways between zebrafish and mice, in addition to the dose‑dependent growth curves of zebrafish embryos upon rapamycin exposure.

  11. Genetic determinants of hyaloid and retinal vasculature in zebrafish

    PubMed Central

    Alvarez, Yolanda; Cederlund, Maria L; Cottell, David C; Bill, Brent R; Ekker, Stephen C; Torres-Vazquez, Jesus; Weinstein, Brant M; Hyde, David R; Vihtelic, Thomas S; Kennedy, Breandan N

    2007-01-01

    Background The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish. Results We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development. Conclusion Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease. PMID:17937808

  12. Sight of conspecific images induces changes in neurochemistry in zebrafish

    PubMed Central

    Saif, Muhhamed; Chatterjee, Diptendu; Buske, Christine; Gerlai, Robert

    2013-01-01

    Zebrafish is gaining popularity in behavioural brain research as this species combines practical simplicity with system complexity. The dopaminergic system has been thoroughly investigated using mammals. Dopamine plays important roles in motor function and reward. Zebrafish have dopamine receptors homologous to mammalian counterparts, and dopamine receptor antagonists as well as alcohol have been shown to exert significant effects on this species as measured using HPLC or behavioural methods. The sight of conspecifics was previously shown to be rewarding in zebrafish but whether this stimulus affects the dopaminergic system has not been studied. Here, we present animated images of zebrafish to the experimental zebrafish subject for varying lengths of time and quantify the amount of dopamine, DOPAC, serotonin and 5HIAA extracted from the subject's brain immediately after the stimulus presentation using HPLC with electrochemical detection. We find conspecific images to induce a robust behavioural response (attraction) in experimental zebrafish. Importantly, dopamine and DOPAC levels significantly increased in response to the presentation of conspecific images but not to scrambled images. Last, serotonin and 5HIAA levels did not significantly change in response to the conspecific images. We conclude that our findings, together with pervious studies, now conclusively demonstrate that the behavioural response induced by the appearance of conspecifics is mediated, at least partly, by the dopaminergic system in zebrafish. PMID:23357085

  13. Sight of conspecific images induces changes in neurochemistry in zebrafish.

    PubMed

    Saif, Muhammed; Chatterjee, Diptendu; Buske, Christine; Gerlai, Robert

    2013-04-15

    Zebrafish are gaining popularity in behavioural brain research as this species combines practical simplicity with system complexity. The dopaminergic system has been thoroughly investigated using mammals. Dopamine plays important roles in motor function and reward. Zebrafish have dopamine receptors homologous to mammalian counterparts, and dopamine receptor antagonists as well as alcohol have been shown to exert significant effects on this species as measured using HPLC or behavioural methods. The sight of conspecifics was previously shown to be rewarding in zebrafish but whether this stimulus affects the dopaminergic system has not been studied. Here, we present animated images of zebrafish to the experimental zebrafish subject for varying lengths of time and quantify the amount of dopamine, DOPAC, serotonin and 5HIAA extracted from the subject's brain immediately after the stimulus presentation using HPLC with electrochemical detection. We find conspecific images to induce a robust behavioural response (attraction) in experimental zebrafish. Importantly, dopamine and D