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1.
J Cereb Blood Flow Metab ; 40(2): 298-313, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30398083

RESUMO

Neurovascular coupling (through which local cerebral blood flow changes in response to neural activation are mediated) is impaired in many diseases including diabetes. Current preclinical rodent models of neurovascular coupling rely on invasive surgery and instrumentation, but transgenic zebrafish coupled with advances in imaging techniques allow non-invasive quantification of cerebrovascular anatomy, neural activation, and cerebral vessel haemodynamics. We therefore established a novel non-invasive, non-anaesthetised zebrafish larval model of neurovascular coupling, in which visual stimulus evokes neuronal activation in the optic tectum that is associated with a specific increase in red blood cell speed in tectal blood vessels. We applied this model to the examination of the effect of glucose exposure on cerebrovascular patterning and neurovascular coupling. We found that chronic exposure of zebrafish to glucose impaired tectal blood vessel patterning and neurovascular coupling. The nitric oxide donor sodium nitroprusside rescued all these adverse effects of glucose exposure on cerebrovascular patterning and function. Our results establish the first non-mammalian model of neurovascular coupling, offering the potential to perform more rapid genetic modifications and high-throughput screening than is currently possible using rodents. Furthermore, using this zebrafish model, we reveal a potential strategy to ameliorate the effects of hyperglycemia on cerebrovascular function.

2.
EMBO Rep ; 20(8): e47047, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31379129

RESUMO

We identify a novel endothelial membrane behaviour in transgenic zebrafish. Cerebral blood vessels extrude large transient spherical structures that persist for an average of 23 min before regressing into the parent vessel. We term these structures "kugeln", after the German for sphere. Kugeln are only observed arising from the cerebral vessels and are present as late as 28 days post fertilization. Kugeln do not communicate with the vessel lumen and can form in the absence of blood flow. They contain little or no cytoplasm, but the majority are highly positive for nitric oxide reactivity. Kugeln do not interact with brain lymphatic endothelial cells (BLECs) and can form in their absence, nor do they perform a scavenging role or interact with macrophages. Inhibition of actin polymerization, Myosin II, or Notch signalling reduces kugel formation, while inhibition of VEGF or Wnt dysregulation (either inhibition or activation) increases kugel formation. Kugeln represent a novel Notch-dependent NO-containing endothelial organelle restricted to the cerebral vessels, of currently unknown function.

3.
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
4.
Dev Dyn ; 248(6): 410-425, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30980582

RESUMO

BACKGROUND: Cilia are essential for morphogenesis and maintenance of many tissues. Loss-of-function of cilia in early Zebrafish development causes a range of vascular defects, including cerebral hemorrhage and reduced arterial vascular mural cell coverage. In contrast, loss of endothelial cilia in mice has little effect on vascular development. We therefore used a conditional rescue approach to induce endothelial cilia ablation after early embryonic development and examined the effect on vascular development and mural cell development in postembryonic, juvenile, and adult Zebrafish. RESULTS: ift54(elipsa)-mutant Zebrafish are unable to form cilia. We rescued cilia formation and ameliorated the phenotype of ift54 mutants using a novel Tg(ubi:loxP-ift54-loxP-myr-mcherry,myl7:EGFP)sh488 transgene expressing wild-type ift54 flanked by recombinase sites, then used a Tg(kdrl:cre)s898 transgene to induce endothelial-specific inactivation of ift54 at postembryonic ages. Fish without endothelial ift54 function could survive to adulthood and exhibited no vascular defects. Endothelial inactivation of ift54 did not affect development of tagln-positive vascular mural cells around either the aorta or the caudal fin vessels, or formation of vessels after tail fin resection in adult animals. CONCLUSIONS: Endothelial cilia are not essential for development and remodeling of the vasculature in juvenile and adult Zebrafish when inactivated after embryogenesis.

5.
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
6.
Nat Commun ; 10(1): 453, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30692543

RESUMO

Venous endothelial cells are molecularly and functionally distinct from their arterial counterparts. Although veins are often considered the default endothelial state, genetic manipulations can modulate both acquisition and loss of venous fate, suggesting that venous identity is the result of active transcriptional regulation. However, little is known about this process. Here we show that BMP signalling controls venous identity via the ALK3/BMPR1A receptor and SMAD1/SMAD5. Perturbations to TGF-ß and BMP signalling in mice and zebrafish result in aberrant vein formation and loss of expression of the venous-specific gene Ephb4, with no effect on arterial identity. Analysis of a venous endothelium-specific enhancer for Ephb4 shows enriched binding of SMAD1/5 and a requirement for SMAD binding motifs. Further, our results demonstrate that BMP/SMAD-mediated Ephb4 expression requires the venous-enriched BMP type I receptor ALK3/BMPR1A. Together, our analysis demonstrates a requirement for BMP signalling in the establishment of Ephb4 expression and the venous vasculature.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Proteínas Morfogenéticas Ósseas/genética , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais/genética , Veias/metabolismo , Animais , Animais Geneticamente Modificados , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Células Endoteliais/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Receptor EphB4/genética , Receptor EphB4/metabolismo , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Veias/embriologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
7.
Blood Adv ; 2(20): 2589-2606, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30309860

RESUMO

The transcriptional repressors Gfi1(a) and Gfi1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, Gfi1 and Gfi1b restrict or promote cell proliferation, prevent apoptosis, influence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation. In zebrafish, prRBCs express 2 of 3 zebrafish Gfi1/1b paralogs, Gfi1aa and Gfi1b. The recently identified zebrafish gfi1aa gene trap allele qmc551 drives erythroid green fluorescent protein (GFP) instead of Gfi1aa expression, yet homozygous carriers have normal prRBCs. prRBCs display a maturation defect only after splice morpholino-mediated knockdown of Gfi1b in gfi1aa qmc551 homozygous embryos. To study the transcriptome of the Gfi1aa/1b double-depleted cells, we performed an RNA-Seq experiment on GFP-positive prRBCs sorted from 20-hour-old embryos that were heterozygous or homozygous for gfi1aa qmc551 , as well as wt or morphant for gfi1b We subsequently confirmed and extended these data in whole-mount in situ hybridization experiments on newly generated single- and double-mutant embryos. Combined, the data showed that in the absence of Gfi1aa, the synchronously developing prRBCs were delayed in activating late erythroid differentiation, as they struggled to suppress early erythroid and endothelial transcription programs. The latter highlighted the bipotent nature of the progenitors from which prRBCs arise. In the absence of Gfi1aa, Gfi1b promoted erythroid differentiation as stepwise loss of wt gfi1b copies progressively delayed Gfi1aa-depleted prRBCs even further, showing that Gfi1aa and Gfi1b together set the pace for prRBC differentiation from hemangioblasts.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Eritroblastos/metabolismo , Hemangioblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Peixe-Zebra
8.
J Mol Cell Cardiol ; 109: 38-47, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28684310

RESUMO

AIMS: Ischaemic cardiovascular disease is a major cause of morbidity and mortality worldwide. Despite promising results from pre-clinical animal models, VEGF-based strategies for therapeutic angiogenesis have yet to achieve successful reperfusion of ischaemic tissues in patients. Failure to restore efficient VEGF activity in the ischaemic organ remains a major problem in current pro-angiogenic therapeutic approaches. Plasma membrane calcium ATPase 4 (PMCA4) negatively regulates VEGF-activated angiogenesis via inhibition of the calcineurin/NFAT signalling pathway. PMCA4 activity is inhibited by the small molecule aurintricarboxylic acid (ATA). We hypothesize that inhibition of PMCA4 with ATA might enhance VEGF-induced angiogenesis. METHODS AND RESULTS: We show that inhibition of PMCA4 with ATA in endothelial cells triggers a marked increase in VEGF-activated calcineurin/NFAT signalling that translates into a strong increase in endothelial cell motility and blood vessel formation. ATA enhances VEGF-induced calcineurin signalling by disrupting the interaction between PMCA4 and calcineurin at the endothelial-cell membrane. ATA concentrations at the nanomolar range, that efficiently inhibit PMCA4, had no deleterious effect on endothelial-cell viability or zebrafish embryonic development. However, high ATA concentrations at the micromolar level impaired endothelial cell viability and tubular morphogenesis, and were associated with toxicity in zebrafish embryos. In mice undergoing experimentally-induced hindlimb ischaemia, ATA treatment significantly increased the reperfusion of post-ischaemic limbs. CONCLUSIONS: Our study provides evidence for the therapeutic potential of targeting PMCA4 to improve VEGF-based pro-angiogenic interventions. This goal will require the development of refined, highly selective versions of ATA, or the identification of novel PMCA4 inhibitors.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Ácido Aurintricarboxílico/farmacologia , ATPases Transportadoras de Cálcio/genética , Membrana Celular/genética , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/efeitos dos fármacos , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
9.
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
10.
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
11.
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
12.
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
13.
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 , Proteínas de Membrana , 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
14.
Dev Cell ; 16(6): 909-16, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19531361

RESUMO

Hematopoietic stem cells (HSCs) are first detected in the floor of the embryonic dorsal aorta (DA), and we investigate the signals that induce the HSC program there. We show that while continued Hedgehog (Hh) signaling from the overlying midline structures maintains the arterial program characteristic of the DA roof, a ventral Bmp4 signal induces the blood stem cell program in the DA floor. This patterning of the DA by Hh and Bmp is the mirror image of that in the neural tube, with Hh favoring dorsal rather than ventral cell types, and Bmp favoring ventral rather than dorsal. With the majority of current data supporting a model whereby HSCs derive from arterial endothelium, our data identify the signal driving this conversion. These findings are important for the study of the production of HSCs from embryonic stem cells and establish a paradigm for the development of adult stem cells.


Assuntos
Aorta/citologia , Padronização Corporal , Proteína Morfogenética Óssea 4/metabolismo , Polaridade Celular , Proteínas Hedgehog/metabolismo , Células-Tronco Hematopoéticas/citologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Aorta/embriologia , Artérias/citologia , Artérias/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Proteínas de Peixe-Zebra/genética
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