RESUMO
Vascular endothelial growth factors (VEGFs) control angiogenesis and lymphangiogenesis during development and in pathological conditions. In the zebrafish trunk, Vegfa controls the formation of intersegmental arteries by primary angiogenesis and Vegfc is essential for secondary angiogenesis, giving rise to veins and lymphatics. Vegfd has been largely thought of as dispensable for vascular development in vertebrates. Here, we generated a zebrafish vegfd mutant by genome editing. vegfd mutants display significant defects in facial lymphangiogenesis independent of vegfc function. Strikingly, we find that vegfc and vegfd cooperatively control lymphangiogenesis throughout the embryo, including during the formation of the trunk lymphatic vasculature. Interestingly, we find that vegfd and vegfc also redundantly drive artery hyperbranching phenotypes observed upon depletion of Flt1 or Dll4. Epistasis and biochemical binding assays suggest that, during primary angiogenesis, Vegfd influences these phenotypes through Kdr (Vegfr2) rather than Flt4 (Vegfr3). These data demonstrate that, rather than being dispensable during development, Vegfd plays context-specific indispensable and also compensatory roles during both blood vessel angiogenesis and lymphangiogenesis.
Assuntos
Linfangiogênese/fisiologia , Neovascularização Fisiológica/fisiologia , Fator D de Crescimento do Endotélio Vascular/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Linfangiogênese/genética , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Modelos Biológicos , Mutagênese , Neovascularização Fisiológica/genética , Deleção de Sequência , Transdução de Sinais , Regulação para Cima , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/fisiologia , Fator D de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.
Assuntos
DNA Bacteriano/química , Genoma Bacteriano , Legionella pneumophila/genética , Proteínas de Membrana Transportadoras/genética , Fatores de Virulência/genética , Sequência Conservada , DNA Bacteriano/genética , Microbiologia Ambiental , Humanos , Legionella pneumophila/isolamento & purificação , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Doença dos Legionários/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Virulência/metabolismoRESUMO
Lymphatic vascular development establishes embryonic and adult tissue fluid balance and is integral in disease. In diverse vertebrate organs, lymphatic vessels display organotypic function and develop in an organ-specific manner. In all settings, developmental lymphangiogenesis is considered driven by vascular endothelial growth factor (VEGF) receptor-3 (VEGFR3), whereas a role for VEGFR2 remains to be fully explored. Here, we define the zebrafish Vegf/Vegfr code in receptor binding studies. We find that while Vegfd directs craniofacial lymphangiogenesis, it binds Kdr (a VEGFR2 homolog) but surprisingly, unlike in mammals, does not bind Flt4 (VEGFR3). Epistatic analyses and characterization of a kdr mutant confirm receptor-binding analyses, demonstrating that Kdr is indispensible for rostral craniofacial lymphangiogenesis, but not caudal trunk lymphangiogenesis, in which Flt4 is central. We further demonstrate an unexpected yet essential role for Kdr in inducing lymphatic endothelial cell fate. This work reveals evolutionary divergence in the Vegf/Vegfr code that uncovers spatially restricted mechanisms of developmental lymphangiogenesis.
Assuntos
Células Endoteliais/metabolismo , Evolução Molecular , Linfangiogênese , Fator C de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Ligantes , Camundongos , Ligação Proteica , Proteólise , Reprodutibilidade dos Testes , Fator C de Crescimento do Endotélio Vascular/química , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismoRESUMO
The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.