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1.
FASEB J ; 38(19): e70086, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-39360639

RESUMO

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.


Assuntos
Morfogênese , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Visão Ocular/fisiologia , Visão Ocular/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Retina/metabolismo , Mutação
2.
J Pediatr ; 220: 73-79.e3, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32089332

RESUMO

OBJECTIVE: To assess differences in regional brain temperatures during whole-body hypothermia and test the hypothesis that brain temperature profile is nonhomogenous in infants with hypoxic-ischemic encephalopathy. STUDY DESIGN: Infants with hypoxic-ischemic encephalopathy were enrolled prospectively in this observational study. Magnetic resonance (MR) spectra of basal ganglia, thalamus, cortical gray matter, and white matter (WM) were acquired during therapeutic hypothermia. Regional brain tissue temperatures were calculated from the chemical shift difference between water signal and metabolites in the MR spectra after performing calibration measurements. Overall difference in regional temperature was analyzed by mixed-effects model; temperature among different patterns and severity of injury on MR imaging also was analyzed. Correlation between temperature and depth of brain structure was analyzed using repeated-measures correlation. RESULTS: In total, 53 infants were enrolled (31 girls, mean gestational age: 38.6 ± 2 weeks; mean birth weight: 3243 ± 613 g). MR spectroscopy was acquired at mean age of 2.2 ± 0.6 days. A total of 201 MR spectra were included in the analysis. The thalamus, the deepest structure (36.4 ± 2.3 mm from skull surface), was lowest in temperature (33.2 ± 0.8°C, compared with basal ganglia: 33.5 ± 0.9°C; gray matter: 33.6 ± 0.7°C; WM: 33.8 ± 0.9°C, all P < .001). Temperatures in more superficial gray matter and WM regions (depth: 21.9 ± 2.4 and 21.5 ± 2.2 mm) were greater than the rectal temperatures (33.4 ± 0.4°C, P < .03). There was a negative correlation between temperature and depth of brain structure (rrm = -0.36, P < .001). CONCLUSIONS: Whole-body hypothermia was effective in cooling deep brain structures, whereas superficial structures were warmer, with temperatures significantly greater than rectal temperatures.


Assuntos
Temperatura Corporal/fisiologia , Encéfalo/diagnóstico por imagem , Hipotermia Induzida , Hipóxia-Isquemia Encefálica/terapia , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Encéfalo/fisiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Prospectivos , Reto/fisiologia , Termometria
3.
Development ; 138(9): 1783-94, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21447554

RESUMO

The development of the different muscles within the somite is a complex process that involves the Hedgehog (Hh) signaling pathway. To specify the proper number of muscle cells and organize them spatially and temporally, the Hh signaling pathway needs to be precisely regulated at different levels, but only a few factors external to the pathway have been described. Here, we report for the first time the role of the STAR family RNA-binding protein Quaking A (QkA) in somite muscle development. We show in zebrafish that the loss of QkA function affects fast muscle fiber maturation as well as Hh-induced muscle derivative specification and/or morphogenesis. Mosaic analysis reveals that fast fiber maturation depends on the activity of QkA in the environment of fast fiber progenitors. We further show that Hh signaling requires QkA activity for muscle development. By an in silico approach, we screened the 3'UTRs of known Hh signaling component mRNAs for the Quaking response element and found the transcription factor Gli2a, a known regulator of muscle fate development. Using destabilized GFP as a reporter, we show that the gli2a mRNA 3'UTR is a functional QkA target. Consistent with this notion, the loss of QkA function rescued slow muscle fibers in yot mutant embryos, which express a dominant-negative Gli2a isoform. Thus, our results reveal a new mechanism to ensure muscle cell fate diversity by fine-tuning of the Hh signaling pathway via RNA-binding proteins.


Assuntos
Proteínas Hedgehog/fisiologia , Desenvolvimento Muscular/genética , Proteínas de Ligação a RNA/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Padronização Corporal/fisiologia , Mapeamento Cromossômico , Embrião não Mamífero , Genes Recessivos , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Morfogênese/genética , Morfogênese/fisiologia , Desenvolvimento Muscular/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/fisiologia , Mutação/fisiologia , Proteínas de Ligação a RNA/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética
4.
Development ; 138(6): 1173-81, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21307094

RESUMO

Arterial and venous endothelial cells exhibit distinct molecular characteristics at early developmental stages. These lineage-specific molecular programs are instructive to the development of distinct vascular architectures and physiological conditions of arteries and veins, but their roles in angiogenesis remain unexplored. Here, we show that the caudal vein plexus in zebrafish forms by endothelial cell sprouting, migration and anastomosis, providing a venous-specific angiogenesis model. Using this model, we have identified a novel compound, aplexone, which effectively suppresses venous, but not arterial, angiogenesis. Multiple lines of evidence indicate that aplexone differentially regulates arteriovenous angiogenesis by targeting the HMG-CoA reductase (HMGCR) pathway. Treatment with aplexone affects the transcription of enzymes in the HMGCR pathway and reduces cellular cholesterol levels. Injecting mevalonate, a metabolic product of HMGCR, reverses the inhibitory effect of aplexone on venous angiogenesis. In addition, aplexone treatment inhibits protein prenylation and blocking the activity of geranylgeranyl transferase induces a venous angiogenesis phenotype resembling that observed in aplexone-treated embryos. Furthermore, endothelial cells of venous origin have higher levels of proteins requiring geranylgeranylation than arterial endothelial cells and inhibiting the activity of Rac or Rho kinase effectively reduces the migration of venous, but not arterial, endothelial cells. Taken together, our findings indicate that angiogenesis is differentially regulated by the HMGCR pathway via an arteriovenous-dependent requirement for protein prenylation in zebrafish and human endothelial cells.


Assuntos
Artérias/efeitos dos fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neovascularização Fisiológica/efeitos dos fármacos , Sulfonamidas/farmacologia , Veias/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Animais , Animais Geneticamente Modificados , Artérias/fisiologia , Células Cultivadas , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Embrião não Mamífero , Humanos , Terapia de Alvo Molecular , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Especificidade por Substrato/efeitos dos fármacos , Veias/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia
6.
Development ; 136(19): 3367-76, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19736328

RESUMO

The skeletal muscle basement membrane fulfils several crucial functions during development and in the mature myotome and defects in its composition underlie certain forms of muscular dystrophy. A major component of this extracellular structure is the laminin polymer, which assembles into a resilient meshwork that protects the sarcolemma during contraction. Here we describe a zebrafish mutant, softy, which displays severe embryonic muscle degeneration as a result of initial basement membrane failure. The softy phenotype is caused by a mutation in the lamb2 gene, identifying laminin beta2 as an essential component of this basement membrane. Uniquely, softy homozygotes are able to recover and survive to adulthood despite the loss of myofibre adhesion. We identify the formation of ectopic, stable basement membrane attachments as a novel means by which detached fibres are able to maintain viability. This demonstration of a muscular dystrophy model possessing innate fibre viability following muscle detachment suggests basement membrane augmentation as a therapeutic strategy to inhibit myofibre loss.


Assuntos
Laminina/genética , Laminina/fisiologia , Distrofia Muscular Animal/embriologia , Distrofia Muscular Animal/genética , Mutação , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Membrana Basal/patologia , Sobrevivência Celular , Primers do DNA/genética , Olho/embriologia , Homozigoto , Dados de Sequência Molecular , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular Animal/patologia , Sarcolema/patologia , Homologia de Sequência de Aminoácidos
7.
PLoS Genet ; 4(3): e1000026, 2008 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-18369445

RESUMO

A fundamental problem in developmental biology concerns how multipotent precursors choose specific fates. Neural crest cells (NCCs) are multipotent, yet the mechanisms driving specific fate choices remain incompletely understood. Sox10 is required for specification of neural cells and melanocytes from NCCs. Like sox10 mutants, zebrafish shady mutants lack iridophores; we have proposed that sox10 and shady are required for iridophore specification from NCCs. We show using diverse approaches that shady encodes zebrafish leukocyte tyrosine kinase (Ltk). Cell transplantation studies show that Ltk acts cell-autonomously within the iridophore lineage. Consistent with this, ltk is expressed in a subset of NCCs, before becoming restricted to the iridophore lineage. Marker analysis reveals a primary defect in iridophore specification in ltk mutants. We saw no evidence for a fate-shift of neural crest cells into other pigment cell fates and some NCCs were subsequently lost by apoptosis. These features are also characteristic of the neural crest cell phenotype in sox10 mutants, leading us to examine iridophores in sox10 mutants. As expected, sox10 mutants largely lacked iridophore markers at late stages. In addition, sox10 mutants unexpectedly showed more ltk-expressing cells than wild-type siblings. These cells remained in a premigratory position and expressed sox10 but not the earliest neural crest markers and may represent multipotent, but partially-restricted, progenitors. In summary, we have discovered a novel signalling pathway in NCC development and demonstrate fate specification of iridophores as the first identified role for Ltk.


Assuntos
Proteínas Tirosina Quinases/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Alelos , Animais , Apoptose/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Mapeamento Cromossômico , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Leucócitos/enzimologia , Melanócitos/citologia , Melanócitos/enzimologia , Modelos Biológicos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/enzimologia , Mutação , Crista Neural/citologia , Crista Neural/embriologia , Crista Neural/enzimologia , Filogenia , Proteínas Tirosina Quinases/genética , Fatores de Transcrição SOXE , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
8.
Dev Biol ; 325(2): 329-40, 2009 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19014929

RESUMO

Two hallmarks of vertebrate epimorphic regeneration are a significant increase in the proliferation of normally quiescent cells and a re-activation of genes that are active during embryonic development. It is unclear what the molecular determinants are that regulate these events and how they are coordinated. Zebrafish have the ability to regenerate several compound structures by regulating cell proliferation and gene transcription. We report that fam53b/simplet (smp) regulates both cell proliferation and the transcription of specific genes. In situ hybridization and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in strong up-regulation of the smp gene. In regenerating adult fin, smp expression remained strong in the distal mesenchyme which later expanded to the basal layers of the distal epidermis and distal tip epithelium. Morpholino knockdown of smp reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone H3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, as well as the later formation of ectopic bone. Taken together, these data indicate a requirement for smp in fin regeneration through control of cell proliferation, the regulation of specific genes and proper bone patterning.


Assuntos
Proliferação de Células , Extremidades/fisiologia , Fatores de Transcrição/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Epiderme/crescimento & desenvolvimento , Epiderme/fisiologia , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica , Mesoderma/crescimento & desenvolvimento , Mesoderma/fisiologia , Miocárdio/metabolismo , Osteogênese/fisiologia , Regeneração , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética
9.
Dev Cell ; 8(4): 575-86, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15809039

RESUMO

Somitogenesis is the process by which the segmented precursors of the skeletal muscle and vertebral column are generated during vertebrate embryogenesis. While somitogenesis appears to be a serially homologous, reiterative process, we find that there are differences between the genetic control of early/anterior and late/posterior somitogenesis. We demonstrate that point mutations can cause segmentation defects in either the anterior, middle, or posterior somites in the zebrafish. We find that mutations in zebrafish integrinalpha5 disrupt anterior somite formation, giving a phenotype complementary to the posterior defects seen in the notch pathway mutants after eight/deltaD and deadly seven/notch1a. Double mutants between the notch pathway and integrinalpha5 display somite defects along the entire body axis, with a complete loss of the mesenchymal-to-epithelial transition and Fibronectin matrix assembly in the posterior. Our data suggest that notch- and integrinalpha5-dependent cell polarization and Fibronectin matrix assembly occur concomitantly and interdependently during border morphogenesis.


Assuntos
Padronização Corporal , Integrina alfa5/metabolismo , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Somitos/fisiologia , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Polaridade Celular , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ , Integrina alfa5/genética , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Dados de Sequência Molecular , Morfogênese , Fenótipo , Mutação Puntual , Receptores Notch , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Somitos/citologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
10.
Nature ; 428(6986): 955-9, 2004 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-15057246

RESUMO

Hair cells have highly organized bundles of apical projections, or stereocilia, that are deflected by sound and movement. Displacement of stereocilia stretches linkages at the tips of stereocilia that are thought to gate mechanosensory channels. To identify the molecular machinery that mediates mechanotransduction in hair cells, zebrafish mutants were identified with defects in balance and hearing. In sputnik mutants, stereociliary bundles are splayed to various degrees, with individuals displaying reduced or absent mechanotransduction. Here we show that the defects in sputnik mutants are caused by mutations in cadherin 23 (cdh23). Mutations in Cdh23 also cause deafness and vestibular defects in mice and humans, and the protein is present in hair bundles. We show that zebrafish Cdh23 protein is concentrated near the tips of hair bundles, and that tip links are absent in homozygous sputnik(tc317e) larvae. Moreover, tip links are absent in larvae carrying weak alleles of cdh23 that affect mechanotransduction but not hair bundle integrity. We conclude that Cdh23 is an essential tip link component required for hair-cell mechanotransduction.


Assuntos
Caderinas/metabolismo , Células Ciliadas Auditivas/metabolismo , Mutação/genética , Peixe-Zebra/fisiologia , Alelos , Animais , Caderinas/genética , Cílios/metabolismo , Cílios/ultraestrutura , Perfilação da Expressão Gênica , Células Ciliadas Auditivas/ultraestrutura , Audição/genética , Audição/fisiologia , Larva/genética , Larva/fisiologia , Mecanotransdução Celular/genética , Mecanotransdução Celular/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra
11.
PLoS One ; 14(1): e0211073, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30695021

RESUMO

Through forward genetic screening for mutations affecting visual system development, we identified prominent coloboma and cell-autonomous retinal neuron differentiation, lamination and retinal axon projection defects in eisspalte (ele) mutant zebrafish. Additional axonal deficits were present, most notably at midline axon commissures. Genetic mapping and cloning of the ele mutation showed that the affected gene is slbp, which encodes a conserved RNA stem-loop binding protein involved in replication dependent histone mRNA metabolism. Cells throughout the central nervous system remained in the cell cycle in ele mutant embryos at stages when, and locations where, post-mitotic cells have differentiated in wild-type siblings. Indeed, RNAseq analysis showed down-regulation of many genes associated with neuronal differentiation. This was coincident with changes in the levels and spatial localisation of expression of various genes implicated, for instance, in axon guidance, that likely underlie specific ele phenotypes. These results suggest that many of the cell and tissue specific phenotypes in ele mutant embryos are secondary to altered expression of modules of developmental regulatory genes that characterise, or promote transitions in, cell state and require the correct function of Slbp-dependent histone and chromatin regulatory genes.


Assuntos
Animais Geneticamente Modificados , Orientação de Axônios/genética , Diferenciação Celular , Proliferação de Células , Coloboma , Doenças Retinianas , Proteínas de Peixe-Zebra/deficiência , Peixe-Zebra , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Coloboma/embriologia , Coloboma/genética , Coloboma/patologia , Histonas/genética , Histonas/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Doenças Retinianas/embriologia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética
12.
Neuron ; 44(6): 947-60, 2004 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-15603738

RESUMO

Retinal ganglion cell (RGC) axons are topographically ordered in the optic tract according to their retinal origin. In zebrafish dackel (dak) and boxer (box) mutants, some dorsal RGC axons missort in the optic tract but innervate the tectum topographically. Molecular cloning reveals that dak and box encode ext2 and extl3, glycosyltransferases implicated in heparan sulfate (HS) biosynthesis. Both genes are required for HS synthesis, as shown by biochemical and immunohistochemical analysis, and are expressed maternally and then ubiquitously, likely playing permissive roles. Missorting in box can be rescued by overexpression of extl3. dak;box double mutants show synthetic pathfinding phenotypes that phenocopy robo2 mutants, suggesting that Robo2 function requires HS in vivo; however, tract sorting does not require Robo function, since it is normal in robo2 null mutants. This genetic evidence that heparan sulfate proteoglycan function is required for optic tract sorting provides clues to begin understanding the underlying molecular mechanisms.


Assuntos
Axônios/metabolismo , Proteoglicanas de Heparan Sulfato/biossíntese , N-Acetilglucosaminiltransferases/fisiologia , Vias Visuais/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Proteoglicanas de Heparan Sulfato/genética , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/biossíntese , N-Acetilglucosaminiltransferases/genética , Células Ganglionares da Retina/metabolismo , Vias Visuais/embriologia , Peixe-Zebra
13.
PLoS One ; 13(11): e0207747, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30458023

RESUMO

In zebrafish, the gene choline acetyltransferase a (chata) encodes one of the two ChAT orthologs responsible for the synthesis of acetylcholine. Acetylcholine (ACh) is essential for neuromuscular transmission and its impaired synthesis by ChAT can lead to neuromuscular junction disorders such as congenital myasthenic syndromes in humans. We have identified a novel mutation in the chata gene of zebrafish, chatatk64, in a collection of uncharacterised ENU-induced mutants. This mutant carries a missense mutation in the codon of a highly conserved serine changing it to an arginine (S102R). This serine is conserved among ChATs from zebrafish, rat, mice and chicken to humans. It resides within the catalytic domain and in the vicinity of the active site of the enzyme. However, it has not been reported so far to be required for enzymatic activity. Modelling of the S102R variant change in the ChAT protein crystal structure suggests that the change affects protein structure and has a direct impact on the catalytic domain of the protein which abolishes embryo motility almost completely.


Assuntos
Domínio Catalítico , Colina O-Acetiltransferase/química , Colina O-Acetiltransferase/genética , Embrião não Mamífero/fisiologia , Movimento , Serina , Peixe-Zebra/embriologia , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Colina O-Acetiltransferase/metabolismo , Estabilidade Enzimática , Humanos , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Peixe-Zebra/genética
14.
BMC Genomics ; 8: 44, 2007 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-17286862

RESUMO

BACKGROUND: Comparative teleost studies are of great interest since they are important in aquaculture and in evolutionary issues. Comparing genomes of fully sequenced model fish species with those of farmed fish species through comparative mapping offers shortcuts for quantitative trait loci (QTL) detections and for studying genome evolution through the identification of regions of conserved synteny in teleosts. Here a comparative mapping study is presented by radiation hybrid (RH) mapping genes of the gilthead sea bream Sparus aurata, a non-model teleost fish of commercial and evolutionary interest, as it represents the worldwide distributed species-rich family of Sparidae. RESULTS: An additional 74 microsatellite markers and 428 gene-based markers appropriate for comparative mapping studies were mapped on the existing RH map of Sparus aurata. The anchoring of the RH map to the genetic linkage map resulted in 24 groups matching the karyotype of Sparus aurata. Homologous sequences to Tetraodon were identified for 301 of the gene-based markers positioned on the RH map of Sparus aurata. Comparison between Sparus aurata RH groups and Tetraodon chromosomes (karyotype of Tetraodon consists of 21 chromosomes) in this study reveals an unambiguous one-to-one relationship suggesting that three Tetraodon chromosomes correspond to six Sparus aurata radiation hybrid groups. The exploitation of this conserved synteny relationship is furthermore demonstrated by in silico mapping of gilthead sea bream expressed sequence tags (EST) that give a significant similarity hit to Tetraodon. CONCLUSION: The addition of primarily gene-based markers increased substantially the density of the existing RH map and facilitated comparative analysis. The anchoring of this gene-based radiation hybrid map to the genome maps of model species broadened the pool of candidate genes that mainly control growth, disease resistance, sex determination and reversal, reproduction as well as environmental tolerance in this species, all traits of great importance for QTL mapping and marker assisted selection. Furthermore this comparative mapping approach will facilitate to give insights into chromosome evolution and into the genetic make up of the gilthead sea bream.


Assuntos
Etiquetas de Sequências Expressas , Dourada/genética , Tetraodontiformes/genética , Animais , Mapeamento Cromossômico , Sequência Conservada , Repetições de Microssatélites , Mapeamento de Híbridos Radioativos , Especificidade da Espécie
15.
BMC Genomics ; 8: 11, 2007 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-17212827

RESUMO

BACKGROUND: Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers. RESULTS: We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the Tübingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM. CONCLUSION: By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations.


Assuntos
Mapeamento Cromossômico , Repetições de Microssatélites , Mutação , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Feminino , Genoma , Masculino , Mutagênese , Fenótipo
16.
DNA Res ; 14(3): 135-40, 2007 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-17591665

RESUMO

The Medaka is an excellent genetic system for studies of vertebrate development and disease and environmental and evolutionary biology studies. To facilitate the mapping of markers or the cloning of affected genes in Medaka mutants identified by forward-genetic screens, we have established a panel of whole-genome radiation hybrids (RHs) and RH maps for three Medaka chromosomes. RH mapping is useful, since markers to be mapped need not be polymorphic and one can establish the order of markers that are difficult to resolve by genetic mapping owing to low genetic recombination rates. RHs were generated by fusing the irradiated donor, OLF-136 Medaka cell line, with the host B78 mouse melanoma cells. Of 290 initial RH clones, we selected 93 on the basis of high retention of fragments of the Medaka genome to establish a panel that allows genotyping in the 96-well format. RH maps for linkage groups 12, 17, and 22 were generated using 159 markers. The average retention for the three chromosomes was 19% and the average break point frequency was approximately 33 kb/cR. We estimate the potential resolution of the RH panel to be approximately 186 kb, which is high enough for integrating RH data with bacterial artificial chromosome clones. Thus, this first RH panel will be a useful tool for mapping mutated genes in Medaka.


Assuntos
Cromossomos/genética , Oryzias/genética , Mapeamento de Híbridos Radioativos , Animais , Marcadores Genéticos , Genoma/genética
17.
Aquat Toxicol ; 81(4): 355-64, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17292976

RESUMO

The zebrafish (Danio rerio) embryo toxicity test (DarT) is under consideration as an alternative to the acute fish toxicity test. Microscopically visible developmental disorders or death are the endpoints used to report on toxicity in DarT. These endpoints are easily observed. They, however, rarely reveal mechanisms leading to a toxic effect and are relatively insensitive compared to chronic toxic effects. We hypothesized that, by using gene expression profiles as an additional endpoint, it may be possible to increase the sensitivity and predictive value of DarT. Therefore, as a proof of principle, we exposed zebrafish embryos to the reference compound 3,4-dichloroaniline (3,4-DCA) and analyzed gene expression patterns with a 14k oligonucleotide array. Important stress response genes not included in the microarray were additionally quantified by reverse transcriptase polymerase chain reaction. Six genes involved in biotransformation (cyp1a, ahr2), stress response (nfe212, maft, hmox1) and cell cycle control (fzr1) were significantly regulated. With the exception of fzr1, these genes proved to be differentially expressed in post hatch life stages as well. The identified genes point toward an aryl hydrocarbon receptor-mediated response. Differential gene expression in embryos exposed for 48 h was observed at 3,4-DCA concentrations as low as 0.78 microM, which is more than 10-fold below the concentrations that elicited visible toxic effects. Upon exposure for 5 days, differential expression was detected at concentrations as low as 0.22 microM of 3,4-DCA, which was close to the lowest observed effect concentration (0.11 microM) in the 30-day early life stage test. This study therefore indicates that gene expression analysis in DarT is able to reveal mechanistic information and may also be exploited for the development of replacement methods for chronic fish tests.


Assuntos
Determinação de Ponto Final/métodos , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Testes de Toxicidade/métodos , Peixe-Zebra/metabolismo , Compostos de Anilina/toxicidade , Animais , Primers do DNA , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Larva/efeitos dos fármacos , Larva/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Elife ; 62017 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-28826496

RESUMO

Altered Ca2+ handling is often present in diseased hearts undergoing structural remodeling and functional deterioration. However, whether Ca2+ directly regulates sarcomere structure has remained elusive. Using a zebrafish ncx1 mutant, we explored the impacts of impaired Ca2+ homeostasis on myofibril integrity. We found that the E3 ubiquitin ligase murf1 is upregulated in ncx1-deficient hearts. Intriguingly, knocking down murf1 activity or inhibiting proteasome activity preserved myofibril integrity, revealing a MuRF1-mediated proteasome degradation mechanism that is activated in response to abnormal Ca2+ homeostasis. Furthermore, we detected an accumulation of the murf1 regulator FoxO in the nuclei of ncx1-deficient cardiomyocytes. Overexpression of FoxO in wild type cardiomyocytes induced murf1 expression and caused myofibril disarray, whereas inhibiting Calcineurin activity attenuated FoxO-mediated murf1 expression and protected sarcomeres from degradation in ncx1-deficient hearts. Together, our findings reveal a novel mechanism by which Ca2+ overload disrupts myofibril integrity by activating a Calcineurin-FoxO-MuRF1-proteosome signaling pathway.


Assuntos
Calcineurina/genética , Cálcio/metabolismo , Proteína Forkhead Box O1/genética , Miócitos Cardíacos/metabolismo , Miofibrilas/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Calcineurina/metabolismo , Sinalização do Cálcio , Embrião não Mamífero , Proteína Forkhead Box O1/metabolismo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Miofibrilas/ultraestrutura , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
19.
Zebrafish ; 14(6): 547-551, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28968196

RESUMO

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.


Assuntos
Bem-Estar do Animal , Eutanásia Animal , Peixe-Zebra/fisiologia , Anestesia/veterinária , Animais , Ciência dos Animais de Laboratório/educação
20.
J Neurosci ; 25(28): 6610-20, 2005 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-16014722

RESUMO

shocked (sho) is a zebrafish mutation that causes motor deficits attributable to CNS defects during the first2dof development. Mutant embryos display reduced spontaneous coiling of the trunk, diminished escape responses when touched, and an absence of swimming. A missense mutation in the slc6a9 gene that encodes a glycine transporter (GlyT1) was identified as the cause of the sho phenotype. Antisense knock-down of GlyT1 in wild-type embryos phenocopies sho, and injection of wild-type GlyT1 mRNA into mutants rescues them. A comparison of glycine-evoked inward currents in Xenopus oocytes expressing either the wild-type or mutant protein found that the missense mutation results in a nonfunctional transporter. glyt1 and the related glyt2 mRNAs are expressed in the hindbrain and spinal cord in nonoverlapping patterns. The fact that these regions are known to be required for generation of early locomotory behaviors suggests that the regulation of extracellular glycine levels in the CNS is important for proper function of neural networks. Furthermore, physiological analysis after manipulation of glycinergic activity in wild-type and sho embryos suggests that the mutant phenotype is attributable to elevated extracellular glycine within the CNS.


Assuntos
Sistema Nervoso Central/embriologia , Proteínas da Membrana Plasmática de Transporte de Glicina/fisiologia , Glicina/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/genética , Animais , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/patologia , Embrião não Mamífero/fisiopatologia , Líquido Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Proteínas da Membrana Plasmática de Transporte de Glicina/antagonistas & inibidores , Proteínas da Membrana Plasmática de Transporte de Glicina/deficiência , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Músculos/embriologia , Músculos/fisiologia , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Oócitos , Fenótipo , Estimulação Física , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Sarcosina/análogos & derivados , Sarcosina/farmacologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Natação , Xenopus , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
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