RESUMO
Endothelial tubulogenesis is a crucial step in the formation of functional blood vessels during angiogenesis and vasculogenesis. Here, we use in vivo imaging of living zebrafish embryos expressing fluorescent fusion proteins of beta-Actin, alpha-Catenin, and the ERM family member Moesin1 (Moesin a), to define a novel cord hollowing process that occurs during the initial stages of tubulogenesis in intersegmental vessels (ISVs) in the embryo. We show that the primary lumen elongates along cell junctions between at least two endothelial cells during embryonic angiogenesis. Moesin1-EGFP is enriched around structures that resemble intracellular vacuoles, which fuse with the luminal membrane during expansion of the primary lumen. Analysis of silent heart mutant embryos shows that initial lumen formation in the ISVs is not dependent on blood flow; however, stabilization of a newly formed lumen is dependent upon blood flow. Zebrafish moesin1 knockdown and cell transplantation experiments demonstrate that Moesin1 is required in the endothelial cells of the ISVs for in vivo lumen formation. Our analyses suggest that Moesin1 contributes to the maintenance of apical/basal cell polarity of the ISVs as defined by adherens junctions. Knockdown of the adherens junction protein Ve-cadherin disrupts formation of the apical membrane and lumen in a cell-autonomous manner. We suggest that Ve-cadherin and Moesin1 function to establish and maintain apical/basal polarity during multicellular lumen formation in the ISVs.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Embrião não Mamífero/irrigação sanguínea , Embrião não Mamífero/metabolismo , Células Endoteliais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Junções Aderentes/metabolismo , Animais , Antígenos CD/genética , Caderinas/genética , Polaridade Celular , Células Endoteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Neovascularização Fisiológica , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Peixe-Zebra/genética , Proteína da Zônula de Oclusão-1RESUMO
We combined reverse and chemical genetics to identify targets and compounds modulating blood vessel development. Through transcript profiling in mice, we identified 150 potentially druggable microvessel-enriched gene products. Orthologs of 50 of these were knocked down in a reverse genetic screen in zebrafish, demonstrating that 16 were necessary for developmental angiogenesis. In parallel, 1280 pharmacologically active compounds were screened in a human cell-based assay, identifying 28 compounds selectively inhibiting endothelial sprouting. Several links were revealed between the results of the reverse and chemical genetic screens, including the serine/threonine (S/T) phosphatases ppp1ca, ppp1cc, and ppp4c and an inhibitor of this gene family; Endothall. Our results suggest that the combination of reverse and chemical genetic screens, in vertebrates, is an efficient strategy for the identification of drug targets and compounds that modulate complex biological systems, such as angiogenesis.
Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/genética , Neovascularização Fisiológica/efeitos dos fármacos , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Inibidores da Angiogênese/metabolismo , Animais , Células Cultivadas , Ácidos Dicarboxílicos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/citologia , Regulação da Expressão Gênica , Humanos , Camundongos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Peixe-ZebraRESUMO
Transposons are one means that nature has used to introduce new genetic material into chromosomes of organisms from every kingdom. They have been extensively used in prokaryotic and lower eukaryotic systems, but until recently there was no transposon that had significant activity in vertebrates. The Sleeping Beauty (SB) transposon system was developed to direct the integration of precise DNA sequences into chromosomes. The SB system was derived from salmonid sequences that had been inactive for more than 10 million years. SB transposons have been used for two principle uses--as a vector for transgenesis and as a method for introducing various trap vectors into (gene-trap) or in the neighborhood of (enhancer-trap) genes to identify their functions. Results of these studies show that SB-mediated transgenesis is more efficient than that by injection of simple plasmids and that expression of transgenesis is stable and reliable following passage through the germline.
Assuntos
Elementos de DNA Transponíveis/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Genômica/métodos , Salmonidae/genética , Animais , Proteínas de Fluorescência VerdeRESUMO
We have identified the zebrafish homologue of VE-cadherin and documented its expression in the developing vascular system. The zebrafish VE-cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole-mount in situ hybridization with the VE-cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE-cadherin expression to demonstrate a conservation of vascular endothelial growth factor-A (VEGF-A) function. The morpholino antisense oligonucleotide knockdown of VEGF-A function in zebrafish embryos results in a loss of angiogenic blood vessels, as indicated by the lack of VE-cadherin expression in the intersegmental vasculature. This loss can be restored in embryos supplemented with either zebrafish or human VEGF-A, the latter indicating that genes crucial to angiogenesis have highly conserved functional activities in vertebrates.
