RESUMO
The extracellular matrix plays a critical role in neural crest (NC) cell migration. In this study, we characterize the contribution of the novel GPI-linked matrix metalloproteinase (MMP) zebrafish mmp17b. Mmp17b is expressed post-gastrulation in the developing NC. Morpholino inactivation of mmp17b function, or chemical inhibition of MMP activity results in aberrant NC cell migration with minimal change in NC proliferation or apoptosis. Intriguingly, a GPI anchored protein with metalloproteinase inhibitor properties, Reversion-inducing-Cysteine-rich protein with Kazal motifs (RECK), which has previously been implicated in NC development, is expressed in close apposition to NC cells expressing mmp17b, raising the possibility that these two gene products interact. Consistent with this possibility, embryos silenced for mmp17b show defective development of the dorsal root ganglia (DRG), a crest-derived structure affected in RECK mutant fish sensory deprived (sdp). Taken together, this study has identified the first pair of MMP, and their putative MMP inhibitor RECK that functions together in NC cell migration.
Assuntos
Movimento Celular/genética , Metaloproteinase 17 da Matriz/genética , Metaloproteinase 17 da Matriz/metabolismo , Crista Neural/citologia , Crista Neural/metabolismo , Sequência de Aminoácidos , Animais , Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Ativação Enzimática , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Perfilação da Expressão Gênica , Metaloproteinase 17 da Matriz/química , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Alinhamento de Sequência , Peixe-ZebraRESUMO
Endothelial cell-specific chemotaxis receptor (ECSCR) is a cell surface protein expressed by blood endothelial cells with roles in endothelial cell migration and signal transduction. We investigated the function of ecscr in the development of the zebrafish vasculature. Zebrafish ecscr is expressed in angioblasts and in axial vessels during angioblast migration and vasculogenesis. Morpholino-directed ecscr knockdown resulted in defective angioblast migration in the posterior lateral plate mesoderm, a process known to depend on vascular endothelial-derived growth factor (VEGF). In cultured cells, transfected ECSCR localized to actin-rich membrane protrusions, colocalizing with kinase insert domain protein receptor (KDR)/VEGF receptor 2 in these regions. ECSCR-silenced cells show reduced VEGF-induced phosphorylation of KDR but not of FMS-like tyrosine kinase 1 (FLT1)/VEGF receptor 1. Finally, chemical inhibition of VEGF receptor activity in zebrafish resulted in angioblast deficiencies that partially overlap with those seen in ecscr morphants. We propose that ecscr promotes migration of zebrafish angioblasts by enhancing endothelial kdr sensitivity to VEGF.
Assuntos
Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Quimiotaxia/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Sequência de Bases , Movimento Celular , Proliferação de Células , Células Cultivadas , Primers do DNA/genética , Células Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Recently, messenger RNAs in eukaryotes have shown to associate with antisense (AS) transcript partners that are often referred to as long noncoding RNAs (lncRNAs) whose function is largely unknown. Here, we have identified a natural AS transcript for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1), tie-1AS lncRNA in zebrafish, mouse, and humans. In embryonic zebrafish, tie-1AS lncRNA transcript is expressed temporally and spatially in vivo with its native target, the tie-1 coding transcript and in additional locations (ear and brain). The tie-1AS lncRNA selectively binds tie-1 mRNA in vivo and regulates tie-1 transcript levels, resulting in specific defects in endothelial cell contact junctions in vivo and in vitro. The ratio of tie-1 versus tie-1AS lncRNA is altered in human vascular anomaly samples. These results directly implicate noncoding RNA-mediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development.
Assuntos
Loci Gênicos/genética , RNA Antissenso/genética , RNA não Traduzido/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio/efeitos dos fármacos , Endotélio/metabolismo , Endotélio/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Junções Intercelulares/efeitos dos fármacos , Junções Intercelulares/metabolismo , Junções Intercelulares/patologia , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Fenótipo , RNA Antissenso/metabolismo , RNA não Traduzido/metabolismo , Receptor de TIE-1/genética , Receptor de TIE-1/metabolismo , Especificidade da Espécie , Doenças Vasculares/genética , Doenças Vasculares/patologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Proteínas de Peixe-Zebra/genéticaRESUMO
In vertebrates, molecular mechanisms dictate angioblasts' migration and subsequent differentiation into arteries and veins. In this study, we used a microarray screen to identify a novel member of the sucrose nonfermenting related kinase (snrk-1) family of serine/threonine kinases expressed specifically in the embryonic zebrafish vasculature and investigated its function in vivo. Using gain- and loss-of-function studies in vivo, we show that Snrk-1 plays an essential role in the migration, maintenance, and differentiation of angioblasts. The kinase function of Snrk-1 is critical for migration and maintenance, but not for the differentiation of angioblasts. In vitro, snrk-1 knockdown endothelial cells show only defects in migration. The snrk-1 gene acts downstream or parallel to notch and upstream of gridlock during artery-vein specification, and the human gene compensates for zebrafish snrk-1 knockdown, suggesting evolutionary conservation of function.