RESUMO
Transgenic zebrafish have been utilized for in vivo analysis of cell behaviors using advanced imaging techniques, for analyzing spatiotemporal gene regulation, and for targeted mis-expression of transgenes. The Tg(fli1a:EGFP)y1 vascular reporter has been particularly useful for examining the development of blood and lymphatic vessels, but it has been suggested that whole-mount in situ hybridization may result high background staining in this line, potentially limiting its usefulness. Here, we show that off-target hybridization of plasmid vector-derived probes to tissues expressing transgenes occurs in a number of different commonly used transgenic lines as a result of multiple cloning site sequences present in the cloning vectors, suggesting this may be a more general problem. However, we also show that this problem is easily avoided by performing in situ hybridization using probes synthesized from PCR templates lacking vector sequences.
Assuntos
Sondas de DNA , Hibridização In Situ/veterinária , Peixe-Zebra/genética , Animais , Embrião não Mamífero , Hibridização In Situ/métodos , Reação em Cadeia da PolimeraseRESUMO
The zebrafish has emerged as an excellent vertebrate model system for studying blood and lymphatic vascular development. The small size, external and rapid development, and optical transparency of zebrafish embryos are some of the advantages the zebrafish model system offers. Multiple well-established techniques have been developed for imaging and functionally manipulating vascular tissues in zebrafish embryos, expanding on and amplifying these basic advantages and accelerating use of this model system for studying vascular development. In the past decade, studies performed using zebrafish as a model system have provided many novel insights into vascular development. In this article we discuss the amenability of this model system for studying blood vessel development and review contributions made by this system to our understanding of vascular development.
Assuntos
Vasos Sanguíneos/embriologia , Neovascularização Fisiológica/fisiologia , Peixe-Zebra/embriologia , Animais , Encéfalo/irrigação sanguínea , Permeabilidade Capilar/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/embriologia , Rim/irrigação sanguínea , Sistema Linfático/embriologia , Microtúbulos , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Transdução de Sinais/fisiologia , Tubulina (Proteína) , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Here, we show that a novel Rspo1-Wnt-Vegfc-Vegfr3 signaling pathway plays an essential role in developmental angiogenesis. A mutation in R-spondin1 (rspo1), a Wnt signaling regulator, was uncovered during a forward-genetic screen for angiogenesis-deficient mutants in the zebrafish. Embryos lacking rspo1 or the proposed rspo1 receptor kremen form primary vessels by vasculogenesis, but are defective in subsequent angiogenesis. Endothelial cell-autonomous inhibition of canonical Wnt signaling also blocks angiogenesis in vivo. The pro-angiogenic effects of Rspo1/Wnt signaling are mediated by Vegfc/Vegfr3(Flt4) signaling. Vegfc expression is dependent on Rspo1 and Wnt, and Vegfc and Vegfr3 are necessary to promote angiogenesis downstream from Rspo1-Wnt. As all of these molecules are expressed by the endothelium during sprouting stages, these results suggest that Rspo1-Wnt-VegfC-Vegfr3 signaling plays a crucial role as an endothelial-autonomous permissive cue for developmental angiogenesis.
Assuntos
Neovascularização Fisiológica/genética , Fator C de Crescimento do Endotélio Vascular/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Via de Sinalização Wnt/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Células Cultivadas , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Trombospondinas , Regulação para Cima/genética , Regulação para Cima/fisiologia , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
The cranial vasculature is essential for the survival and development of the central nervous system and is important in stroke and other brain pathologies. Cranial vessels form in a reproducible and evolutionarily conserved manner, but the process by which these vessels assemble and acquire their stereotypic patterning remains unclear. Here, we examine the stepwise assembly and patterning of the vascular network of the zebrafish hindbrain. The major artery supplying the hindbrain, the basilar artery, runs along the ventral keel of the hindbrain in all vertebrates. We show that this artery forms by a novel process of medial sprouting and migration of endothelial cells from a bilateral pair of primitive veins, the primordial hindbrain channels. Subsequently, a second wave of dorsal sprouting from the primordial hindbrain channels gives rise to angiogenic central arteries that penetrate into and innervate the hindbrain. The chemokine receptor cxcr4a is expressed in migrating endothelial cells of the primordial hindbrain channels, whereas its ligand cxcl12b is expressed in the hindbrain neural keel immediately adjacent to the assembling basilar artery. Knockdown of either cxcl12b or cxcr4a results in defects in basilar artery formation, showing that the assembly and patterning of this crucial artery depends on chemokine signaling.
