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1.
Nat Commun ; 10(1): 2024, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048699

RESUMO

Mutations in the polycystins cause autosomal dominant polycystic kidney disease (ADPKD). Here we show that transmembrane protein 33 (TMEM33) interacts with the ion channel polycystin-2 (PC2) at the endoplasmic reticulum (ER) membrane, enhancing its opening over the whole physiological calcium range in ER liposomes fused to planar bilayers. Consequently, TMEM33 reduces intracellular calcium content in a PC2-dependent manner, impairs lysosomal calcium refilling, causes cathepsins translocation, inhibition of autophagic flux upon ER stress, as well as sensitization to apoptosis. Invalidation of TMEM33 in the mouse exerts a potent protection against renal ER stress. By contrast, TMEM33 does not influence pkd2-dependent renal cystogenesis in the zebrafish. Together, our results identify a key role for TMEM33 in the regulation of intracellular calcium homeostasis of renal proximal convoluted tubule cells and establish a causal link between TMEM33 and acute kidney injury.


Assuntos
Lesão Renal Aguda/patologia , Cálcio/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas de Membrana/metabolismo , Canais de Cátion TRPP/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Lesão Renal Aguda/genética , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Túbulos Renais Proximais/citologia , Lisossomos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologia
2.
Nat Commun ; 10(1): 732, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760708

RESUMO

Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development.


Assuntos
Sinalização do Cálcio , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Retículo Endoplasmático/metabolismo , MAP Quinases Reguladas por Sinal Extracelular , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Fosforilação , Peixe-Zebra
3.
Proc Natl Acad Sci U S A ; 114(37): 9948-9953, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28851829

RESUMO

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src-mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.


Assuntos
Glucocorticoides/farmacologia , Glucocorticoides/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Hipóxia Celular/fisiologia , Humanos , Hipóxia , Ligases/metabolismo , Fígado/metabolismo , Ligação Proteica , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Peixe-Zebra , Doença de von Hippel-Lindau/metabolismo
4.
PLoS One ; 10(10): e0141611, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26506092

RESUMO

INTRODUCTION AND OBJECTIVES: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development. METHODS AND RESULTS: Using Transcription Activator-Like Effector Nucleases (TALEN) we generated a klf2a mutant (klf2ash317) with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC) development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl), a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17) in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants. CONCLUSIONS: The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.


Assuntos
Sistema Cardiovascular/crescimento & desenvolvimento , Sistema Hematopoético/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/genética , Morfogênese/genética , Proteínas de Peixe-Zebra/genética , Animais , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/biossíntese , Morfolinos/genética , Mutação , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/biossíntese
5.
Prog Mol Biol Transl Sci ; 124: 65-91, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24751427

RESUMO

The zebrafish has been rapidly adopted as a model for cardiac development and disease. The transparency of the embryo, its limited requirement for active oxygen delivery, and ease of use in genetic manipulations and chemical exposure have made it a powerful alternative to rodents. Novel technologies like TALEN/CRISPR-mediated genome engineering and advanced imaging methods will only accelerate its use. Here, we give an overview of heart development and function in the fish and highlight a number of areas where it is most actively contributing to the understanding of cardiac development and disease. We also review the current state of research on a feature that we only could wish to be conserved between fish and human; cardiac regeneration.


Assuntos
Modelos Animais de Doenças , Cardiopatias/patologia , Peixe-Zebra/fisiologia , Animais , Fenômenos Eletrofisiológicos , Coração/embriologia , Coração/fisiologia , Regeneração/fisiologia , Peixe-Zebra/embriologia
6.
Prog Mol Biol Transl Sci ; 124: 93-122, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24751428

RESUMO

The zebrafish has recently emerged as an important animal model to study the formation of the vertebrate vascular network. The small size, optical translucency, and genetic tractability of the zebrafish embryo, in combination with an abundance of fluorescent transgenic lines which permit direct visualization of in vivo vessel formation, have greatly advanced our understanding of vascular biology. Widespread adoption of this powerful system has led to many important discoveries in relation to the mechanisms that underlie blood vessel formation. This review highlights the contribution of the zebrafish system to the current understanding of blood vessel formation and the use of zebrafish to model human vascular disease.


Assuntos
Vasos Sanguíneos/embriologia , Doenças Vasculares/patologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Vasos Sanguíneos/patologia , Modelos Animais de Doenças , Humanos , Linfangiogênese , Desenvolvimento Muscular
7.
PLoS Genet ; 9(12): e1003955, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24339784

RESUMO

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles.


Assuntos
Cílios/genética , Cinesina/genética , Proteínas Oncogênicas/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Anormalidades Múltiplas , Animais , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , Cerebelo/anormalidades , Embrião não Mamífero/metabolismo , Extremidades/crescimento & desenvolvimento , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Cinesina/metabolismo , Camundongos , Tubo Neural/crescimento & desenvolvimento , Proteínas Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Retina/anormalidades , Retina/patologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco
8.
Biotechniques ; 55(6): 314-6, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24344681

RESUMO

Here we describe a method for high-throughput genotyping of live larval zebrafish as early as 72 h post-fertilization (hpf). Importantly, this technique allows rapid and cost-effective PCR-based genotyping from very small fin biopsies, which regenerate as the embryo develops, thereby allowing researchers to select embryos with desired genotypes to be raised to adulthood.


Assuntos
Técnicas de Genotipagem , Peixe-Zebra/genética , Animais , Biópsia/veterinária , Sequenciamento de Nucleotídeos em Larga Escala , Larva/genética , Reação em Cadeia da Polimerase/métodos , Cauda/patologia , Peixe-Zebra/crescimento & desenvolvimento
9.
Cardiovasc Res ; 100(2): 252-61, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23812297

RESUMO

AIMS: The contribution of blood flow to angiogenesis is incompletely understood. We examined the effect of blood flow on Notch signalling in the vasculature of zebrafish embryos, and whether blood flow regulates angiogenesis in zebrafish with constitutively up-regulated hypoxic signalling. METHODS AND RESULTS: Developing zebrafish (Danio rerio) embryos survive via diffusion in the absence of circulation induced by knockdown of cardiac troponin T2 or chemical cardiac cessation. The absence of blood flow increased vascular Notch signalling in 48 h post-fertilization old embryos via up-regulation of the Notch ligand dll4. Despite this, patterning of the intersegmental vessels is not affected by absent blood flow. We therefore examined homozygous vhl mutant zebrafish that have constitutively up-regulated hypoxic signalling. These display excessive and aberrant angiogenesis from 72 h post-fertilization, with significantly increased endothelial number, vessel diameter, and length. The absence of blood flow abolished these effects, though normal vessel patterning was preserved. CONCLUSION: We show that blood flow suppresses vascular Notch signalling via down-regulation of dll4. We have also shown that blood flow is required for angiogenesis in response to hypoxic signalling but is not required for normal vessel patterning. These data indicate important differences in hypoxia-driven vs. developmental angiogenesis.


Assuntos
Circulação Sanguínea/fisiologia , Hipóxia/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Neovascularização Fisiológica , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Animais , Diacetil/análogos & derivados , Diacetil/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas Supressoras de Tumor/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/fisiologia
10.
Cancer Res ; 72(16): 4017-27, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22665266

RESUMO

Hypoxic signaling is a central modulator of cellular physiology in cancer. Core members of oxygen-sensing pathway including the von Hippel-Lindau tumor suppressor protein (pVHL) and the hypoxia inducible factor (HIF) transcription factors have been intensively studied, but improved organismal models might speed advances for both pathobiologic understanding and therapeutic modulation. To study HIF signaling during tumorigenesis and development in zebrafish, we developed a unique in vivo reporter for hypoxia, expressing EGFP driven by prolyl hydroxylase 3 (phd3) promoter/regulatory elements. Modulation of HIF pathway in Tg(phd3::EGFP) embryos showed a specific role for hypoxic signaling in the transgene activation. Zebrafish vhl mutants display a systemic hypoxia response, reflected by strong and ubiquitous transgene expression. In contrast to human VHL patients, heterozygous Vhl mice and vhl zebrafish are not predisposed to cancer. However, upon exposure to dimethylbenzanthracene (DMBA), the vhl heterozygous fish showed an increase in the occurrence of hepatic and intestinal tumors, a subset of which exhibited strong transgene expression, suggesting loss of Vhl function in these tumor cells. Compared with control fish, DMBA-treated vhl heterozygous fish also showed an increase in proliferating cell nuclear antigen-positive renal tubules. Taken together, our findings establish Vhl as a genuine tumor suppressor in zebrafish and offer this model as a tool to noninvasively study VHL and HIF signaling during tumorigenesis and development.


Assuntos
Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Neoplasias Intestinais/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Animais , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Transformação Celular Neoplásica/induzido quimicamente , Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Intestinais/induzido quimicamente , Neoplasias Hepáticas Experimentais/induzido quimicamente , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo
11.
Blood ; 120(2): 477-88, 2012 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-22668851

RESUMO

Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the phenotypes induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1;ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1(+) HSCs.


Assuntos
Proteína Semelhante a Receptor de Calcitonina/metabolismo , Proteínas Hedgehog/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Artérias/embriologia , Artérias/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Mutação , Receptores Patched , Receptor Patched-1 , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Peixe-Zebra/genética
12.
Development ; 137(8): 1361-71, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20223756

RESUMO

In zebrafish, Hedgehog (Hh) signalling from ventral midline structures is necessary and sufficient to specify posterior otic identity. Loss of Hh signalling gives rise to mirror symmetric ears with double anterior character, whereas severe upregulation of Hh signalling leads to double posterior ears. By contrast, in mouse and chick, Hh is predominantly required for dorsoventral otic patterning. Whereas a loss of Hh function in zebrafish does not affect dorsoventral and mediolateral otic patterning, we now show that a gain of Hh signalling activity causes ventromedial otic territories to expand at the expense of dorsolateral domains. In a panel of lines carrying mutations in Hh inhibitor genes, Hh pathway activity is increased throughout the embryo, and dorsolateral otic structures are lost or reduced. Even a modest increase in Hh signalling has consequences for patterning the ear. In ptc1(-/-) and ptc2(-/-) mutant embryos, in which Hh signalling is maximal throughout the embryo, the inner ear is severely ventralised and medialised, in addition to displaying the previously reported double posterior character. Transplantation experiments suggest that the effects of the loss of Hh pathway inhibition on the ear are mediated directly. These new data suggest that Hh signalling must be kept tightly repressed for the correct acquisition of dorsolateral cell fates in the zebrafish otic vesicle, revealing distinct similarities between the roles of Hh signalling in zebrafish and amniote inner ear patterning.


Assuntos
Audição/fisiologia , Transdução de Sinais/fisiologia , Peixe-Zebra/fisiologia , Animais , Anticorpos/análise , Galinhas , Colágeno/genética , Colágeno/imunologia , Primers do DNA , Orelha Interna/anatomia & histologia , Orelha Interna/fisiologia , Embrião não Mamífero/imunologia , Embrião não Mamífero/fisiologia , Hemoglobinas/genética , Hemoglobinas/fisiologia , Camundongos , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Canais Semicirculares/anatomia & histologia , Canais Semicirculares/fisiologia , Especificidade da Espécie , Peixe-Zebra/genética
13.
Development ; 135(22): 3789-99, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18842815

RESUMO

Elucidation of the complete roster of signals required for myocardial specification is crucial to the future of cardiac regenerative medicine. Prior studies have implicated the Hedgehog (Hh) signaling pathway in the regulation of multiple aspects of heart development. However, our understanding of the contribution of Hh signaling to the initial specification of myocardial progenitor cells remains incomplete. Here, we show that Hh signaling promotes cardiomyocyte formation in zebrafish. Reduced Hh signaling creates a cardiomyocyte deficit, and increased Hh signaling creates a surplus. Through fate-mapping, we find that Hh signaling is required at early stages to ensure specification of the proper number of myocardial progenitors. Genetic inducible fate mapping in mouse indicates that myocardial progenitors respond directly to Hh signals, and transplantation experiments in zebrafish demonstrate that Hh signaling acts cell autonomously to promote the contribution of cells to the myocardium. Thus, Hh signaling plays an essential early role in defining the optimal number of cardiomyocytes, making it an attractive target for manipulation of multipotent progenitor cells.


Assuntos
Coração/embriologia , Proteínas Hedgehog/metabolismo , Miocárdio/metabolismo , Transdução de Sinais , Animais , Diferenciação Celular , Proliferação de Células , Células Endoteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/genética , Camundongos , Miocárdio/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
14.
Circulation ; 116(5): 515-25, 2007 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-17646580

RESUMO

BACKGROUND: Extracellular matrix proteins, such as laminins, and endothelial cells are known to influence cardiomyocyte performance; however, the underlying molecular mechanisms remain poorly understood. METHODS AND RESULTS: We used a forward genetic screen in zebrafish to identify novel genes required for myocardial function and were able to identify the lost-contact (loc) mutant, which encodes a nonsense mutation in the integrin-linked kinase (ilk) gene. This loc/ilk mutant is associated with a severe defect in cardiomyocytes and endothelial cells that leads to severe myocardial dysfunction. Additional experiments revealed the epistatic regulation between laminin-alpha4 (Lama4), integrin, and Ilk, which led us to screen for mutations in the human ILK and LAMA4 genes in patients with severe dilated cardiomyopathy. We identified 2 novel amino acid residue-altering mutations (2828C>T [Pro943Leu] and 3217C>T [Arg1073X]) in the integrin-interacting domain of the LAMA4 gene and 1 mutation (785C>T [Ala262Val]) in the ILK gene. Biacore quantitative protein/protein interaction data, which have been used to determine the equilibrium dissociation constants, point to the loss of integrin-binding capacity in case of the Pro943Leu (Kd=5+/-3 micromol/L) and Arg1073X LAMA4 (Kd=1+/-0.2 micromol/L) mutants compared with the wild-type LAMA4 protein (Kd=440+/-20 nmol/L). Additional functional data point to the loss of endothelial cells in affected patients as a direct consequence of the mutant genes, which ultimately leads to heart failure. CONCLUSIONS: This is the first report on mutations in the laminin, integrin, and ILK system in human cardiomyopathy, which has consequences for endothelial cells as well as for cardiomyocytes, thus providing a new genetic basis for dilated cardiomyopathy in humans.


Assuntos
Cardiomiopatia Dilatada/genética , Células Endoteliais/patologia , Laminina/genética , Mutação de Sentido Incorreto , Miócitos Cardíacos/patologia , Mutação Puntual , Proteínas Serina-Treonina Quinases/genética , Adulto , Substituição de Aminoácidos , Animais , Células COS , Cardiomiopatia Dilatada/metabolismo , Cardiomiopatia Dilatada/patologia , Adesão Celular , Cercopithecus aethiops , Mapeamento Cromossômico , Códon sem Sentido , Análise Mutacional de DNA , Embrião não Mamífero/patologia , Epigênese Genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Coração/embriologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/patologia , Humanos , Integrinas/metabolismo , Laminina/fisiologia , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Miocárdio/patologia , Oligonucleotídeos Antissenso/toxicidade , Linhagem , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/fisiologia , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
15.
Genome Res ; 16(4): 491-7, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16533913

RESUMO

Although zebrafish was introduced as a laboratory model organism several decades ago and now serves as a primary model for developmental biology, there is only limited data on its genetic variation. An establishment of a dense polymorphism map becomes a requirement for effective linkage analysis and cloning approaches in zebrafish. By comparing ESTs to whole-genome shotgun data, we predicted >50,000 high-quality candidate SNPs covering the zebrafish genome with average resolution of 41 kbp. We experimentally validated approximately 65% of a randomly sampled subset by genotyping 16 samples from seven commonly used zebrafish strains. The analysis reveals very high nucleotide diversity between zebrafish isolates. Even with the limited number of samples that we genotyped, zebrafish isolates revealed considerable interstrain variation, ranging from 7% (inbred) to 37% (wild-derived) of polymorphic sites being heterozygous. The increased proportion of polymorphic over monomorphic sites results in five times more frequent observation of a three allelic variant compared with human or mouse. Phylogenetic analysis shows that comparisons between even the least divergent strains used in our analysis may provide one informative marker approximately every 500 nucleotides. Furthermore, the number of haplotypes per locus is relatively large, reflecting independent establishment of the different lines from wild isolates. Finally, our results suggest the presence of prominent C-to-U and A-to-I RNA editing events in zebrafish. Overall, the levels and organization of genetic variation between and within commonly used zebrafish strains are markedly different from other laboratory model organisms, which may affect experimental design and interpretation.


Assuntos
Genoma/genética , Polimorfismo de Nucleotídeo Único , Edição de RNA/genética , Peixe-Zebra/genética , Animais , Mapeamento Cromossômico/métodos , Ligação Genética/genética , Marcadores Genéticos/genética , Especificidade da Espécie
16.
PLoS Genet ; 1(2): e19, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16121254

RESUMO

Proliferation is one of the basic processes that control embryogenesis. To identify factors involved in the regulation of proliferation, we performed a zebrafish genetic screen in which we used proliferating cell nuclear antigen (PCNA) expression as a readout. Two mutants, hu418B and hu540A, show increased PCNA expression. Morphologically both mutants resembled the dre (dreumes), uki (ukkie), and lep (leprechaun) mutant class and both are shown to be additional uki alleles. Surprisingly, although an increased size is detected of multiple structures in these mutant embryos, adults become dwarfs. We show that these mutations disrupt repressors of the Hedgehog (Hh) signaling pathway. The dre, uki, and lep loci encode Su(fu) (suppressor of fused), Hip (Hedgehog interacting protein), and Ptc2 (Patched2) proteins, respectively. This class of mutants is therefore unique compared to previously described Hh mutants from zebrafish genetic screens, which mainly show loss of Hh signaling. Furthermore, su(fu) and ptc2 mutants have not been described in vertebrate model systems before. Inhibiting Hh activity by cyclopamine rescues uki and lep mutants and confirms the overactivation of the Hh signaling pathway in these mutants. Triple uki/dre/lep mutants show neither an additive increase in PCNA expression nor enhanced embryonic phenotypes, suggesting that other negative regulators, possibly Ptc1, prevent further activation of the Hh signaling pathway. The effects of increased Hh signaling resulting from the genetic alterations in the uki, dre, and lep mutants differ from phenotypes described as a result of Hh overexpression and therefore provide additional insight into the role of Hh signaling during vertebrate development.


Assuntos
Mutação , Transdução de Sinais/genética , Transativadores/genética , Animais , Tamanho Corporal , Proliferação de Células , Nanismo , Embrião não Mamífero , Proteínas Hedgehog , Antígeno Nuclear de Célula em Proliferação/genética , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
17.
Cell Microbiol ; 5(9): 601-11, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12925130

RESUMO

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28 h-old zebrafish embryos were infected with DsRed-labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild-type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage-like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non-pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage-like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat-killed, wild-type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host.


Assuntos
Embrião não Mamífero/microbiologia , Salmonelose Animal/microbiologia , Salmonella typhimurium/fisiologia , Peixe-Zebra/embriologia , Animais , Contagem de Colônia Microbiana , Embrião não Mamífero/metabolismo , Escherichia coli/fisiologia , Infecções por Escherichia coli/microbiologia , Macrófagos/citologia , Macrófagos/microbiologia , Salmonella typhimurium/citologia , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Fatores de Tempo
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