RESUMEN
Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic disease caused by mutations affecting components of bone morphogenetic protein (BMP)/transforming growth factor-ß (TGF-ß) signaling in endothelial cells. This disorder is characterized by arteriovenous malformations that are prone to rupture, and the ensuing hemorrhages are responsible for iron-deficiency anemia. Along with activin receptor-like kinase (ALK1), mutations in endoglin are associated with the vast majority of HHT cases. In this study, we characterized the zebrafish endoglin locus and demonstrated that it produces two phylogenetically conserved protein isoforms. Functional analysis of a CRISPR/Cas9 zebrafish endoglin mutant revealed that Endoglin deficiency is lethal during the course from juvenile stage to adulthood. Endoglin-deficient zebrafish develop cardiomegaly, resulting in heart failure and hypochromic anemia, which both stem from chronic hypoxia. endoglin mutant zebrafish display structural alterations of the developing gills and underlying vascular network that coincide with hypoxia. Finally, phenylhydrazine treatment demonstrated that lowering hematocrit/blood viscosity alleviates heart failure and enhances the survival of Endoglin-deficient fish. Overall, our data link Endoglin deficiency to heart failure and establish zebrafish as a valuable HHT model.
Asunto(s)
Insuficiencia Cardíaca , Telangiectasia Hemorrágica Hereditaria , Animales , Endoglina/genética , Endoglina/metabolismo , Telangiectasia Hemorrágica Hereditaria/complicaciones , Telangiectasia Hemorrágica Hereditaria/genética , Pez Cebra , Células Endoteliales/metabolismo , Insuficiencia Cardíaca/metabolismo , Receptores de Activinas Tipo II/genéticaRESUMEN
In humans, several members of the CEACAM receptor family have been shown to interact with intestinal pathogens in an inflammatory context. While CEACAMs have long been thought to be only present in mammals, recent studies have identified ceacam genes in other vertebrates, including teleosts. The function of these related genes remains however largely unknown. To gain insight into the function of CEACAM proteins in fish, we undertook the study of a putative member of the family, CEACAMz1, identified in Danio rerio. Sequence analysis of the ceacamz1 gene product predicted a GPI-anchored extracellular protein containing eleven immunoglobulin domains but revealed no evident orthology with human CEACAMs. Using a combination of RT-PCR analyses and in situ hybridization experiments, as well as a fluorescent reporter line, we showed that CEACAMz1 is first expressed in discrete cells on the ventral skin of zebrafish larvae and later on in the developing gills. This distribution remains constant until juvenile stage is reached, at which point CEACAMz1 is almost exclusively expressed in gills. We further observed that at late larval stages, CEACAMz1-expressing cells mostly localize on the afferent side of the branchial filaments and possibly in the inter-lamellar space. Using immunolabelling and 3D-reconstructions, we showed that CEACAMz1 is expressed in cells from the uppermost layer of skin epidermis. These cells are embedded within the keratinocytes pavement and we unambiguously identified them as proton-pump rich ionocytes (HR cells). As the expression of ceacamz1 is turned on concomitantly to that of other known markers of HR cells, we propose that ceacamz1 may serve as a novel marker of mature HR cells from the zebrafish epidermis.
Asunto(s)
Embrión no Mamífero/metabolismo , Epidermis/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Humanos , Queratinocitos/metabolismo , Sistemas de Lectura Abierta/genética , Bombas de Protones/metabolismo , Piel/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin-mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors, the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms [i.e., the repression of endothelial-derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway, and the expression of leukocyte adhesion molecules and of RHOA by endothelial cells], it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar-associated glycoprotein-1 (MAGP-1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Although EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP-1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions.
Asunto(s)
Adhesión Celular , Proteínas Contráctiles/metabolismo , Factores de Crecimiento Endotelial/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Transducción de Señal , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio , Familia de Proteínas EGF , Humanos , Proteína-Lisina 6-Oxidasa/metabolismo , Factores de Empalme de ARN , Receptores Notch/metabolismo , Proteínas Represoras/metabolismoRESUMEN
In vertebrates, haematopoietic stem/progenitor cells (HSPCs) first emerge in the aorta-gonad-mesonephros (AGM) before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life. Here we identify an unexpected primitive macrophage population accumulated in the dorsal mesenteric mesoderm surrounding the dorsal aorta of the human embryo and study its function in the transparent zebrafish embryo. Our study reveals dynamic interactions occurring between the HSPCs and primitive macrophages in the AGM. Specific chemical and inducible genetic depletion of macrophages or inhibition of matrix metalloproteinases (Mmps) leads to an accumulation of HSPCs in the AGM and a decrease in the colonization of haematopoietic organs. Finally, in vivo zymography demonstrates the function of primitive macrophages in extracellular matrix degradation, which allows HSPC migration through the AGM stroma, their intravasation, leading to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis.
Asunto(s)
Aorta/embriología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Macrófagos/citología , Células Madre/citología , Animales , Animales Modificados Genéticamente , Linaje de la Célula , Biología Evolutiva , Matriz Extracelular/metabolismo , Gónadas/embriología , Humanos , Macrófagos/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Mesonefro/embriología , Microscopía Fluorescente , Pez CebraRESUMEN
Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a -8409/-7563 enhancer and the -252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage.
Asunto(s)
Células Endoteliales/metabolismo , Elementos de Facilitación Genéticos/genética , Factor de Transcripción GATA2/metabolismo , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Proteínas/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Proteínas de Unión al Calcio , Cromosomas de los Mamíferos/genética , Familia de Proteínas EGF , Eliminación de Gen , Sitios Genéticos/genética , Humanos , Ratones , Proteínas/metabolismo , Transcripción GenéticaRESUMEN
We previously characterized VE-statin/egfl7, a protein that is exclusively secreted by endothelial cells and modulates smooth muscle cell migration. Here, we show that VE-statin/egfl7 is the first known natural negative regulator of vascular elastogenesis. Transgenic mice, expressing VE-statin/egfl7 under the control of keratin-14 promoter, showed an accumulation of VE-statin/egfl7 in arterial walls where its presence correlated with an impaired organization of elastic fibres. In vitro, fibroblasts cultured in the presence of VE-statin/egfl7 were unable to deposit elastic fibres due to a deficient conversion of soluble tropoelastin into insoluble mature elastin. VE-statin/egfl7 interacts with the catalytic domain of lysyl oxidase (LOX) enzymes and, in endothelial cells, endogenous VE-statin/egfl7 colocalizes with LoxL2 and inhibits elastic fibre deposition. In contrast, mature elastic fibres are abundantly deposited by endothelial cells that are prevented from producing endogenous VE-statin/egfl7. We propose a model where VE-statin/egfl7 produced by endothelial cells binds to the catalytic domains of enzymes of the LOX family in the vascular wall, thereby preventing the crosslink of tropoelastin molecules into mature elastin polymers and regulating vascular elastogenesis.
Asunto(s)
Vasos Sanguíneos/enzimología , Elastina/biosíntesis , Proteína-Lisina 6-Oxidasa/metabolismo , Proteínas/metabolismo , Animales , Vasos Sanguíneos/anomalías , Proteínas de Unión al Calcio , Dominio Catalítico , Línea Celular , Familia de Proteínas EGF , Tejido Elástico/metabolismo , Células Endoteliales/citología , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Humanos , Queratinocitos/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Fenotipo , Unión Proteica , Transporte de Proteínas , Proteína-Lisina 6-Oxidasa/química , Anomalías Cutáneas/patología , Transcripción Genética , Tropoelastina/genéticaRESUMEN
Phosphoinositide 3-kinase (PI3K) is activated by transmembrane tyrosine kinases such as vascular endothelial growth factor (VEGF) receptors and Tie2 (tunica intima endothelial kinase 2), both of which are key regulators of vascular development. However, the in vivo role of PI3K during developmental vascularization remains to be defined. Here we demonstrate that mice deficient in the p110alpha catalytic subunit of PI3K display multiple vascular defects, including dilated vessels in the head, reduced branching morphogenesis in the endocardium, lack of hierarchical order of large and small branches in the yolk sac, and impaired development of anterior cardinal veins. These vascular defects are strikingly similar to those in mice defective in the Tie2 signaling pathway. Indeed, Tie2 protein levels were significantly lower in p110alpha-deficient mice. Furthermore, RNA interference of p110alpha in cultured endothelial cells significantly reduced Tie2 protein levels. These findings raise the possibility that PI3K may function as an upstream regulator of Tie2 expression during mouse development.
Asunto(s)
Embrión de Mamíferos/anomalías , Embrión de Mamíferos/irrigación sanguínea , Regulación del Desarrollo de la Expresión Génica , Fosfatidilinositol 3-Quinasas/deficiencia , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Células Endoteliales/metabolismo , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Subunidades de Proteína/deficiencia , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptor TIE-2/deficienciaRESUMEN
We have identified the mouse exon VII splice variant of the Ets-1 transcription factor. The variant is expressed in all cell lines which express ets-1, at lower levels, it is also expressed in the mouse embryo in vivo. The corresponding protein, p42Ets-1, is a transcription factor as it is able to bind to specific DNA sequences and to transactivate a bona fide ETS reporter vector. A comparison of optimal DNA-binding sites shows that p42Ets-1 binds to more various DNA sequences than p51Ets-1; p42Ets-1 recognizes the same optimal consensus sequence as p51Ets-1, but also many variations of it, mainly at base -1, which is located just prior to the GGAA/T core sequence. The binding differences were quantified by surface plasmon resonance analyses and the protein region responsible for the differences in DNA sequence recognition located in the Val280-Glu302 fragment, which is encoded by exon VII. The specific DNA-binding properties of each isoform translates into clear differences in activity, p42Ets-1 transactivates the natural VE-cadherin gene promoter through both ETS-binding site (EBS)2 and EBS4 whereas p51Ets-1 is mainly active on EBS4. Altogether, our data suggest that p42Ets-1 acts as a distinct transcription factor from p51Ets-1.
Asunto(s)
Empalme Alternativo , Variación Genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , ADN de Neoplasias/química , ADN de Neoplasias/metabolismo , Exones , Vectores Genéticos , Ácido Glutámico , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas Tirosina Quinasas/genética , Proteína Proto-Oncogénica c-ets-1 , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Activación Transcripcional , ValinaRESUMEN
To understand the role of the Ets-1 transcription factor during angiogenesis, we have overexpressed it in endothelial cells and analyzed the levels of expression of several candidate target genes involved in angiogenesis. The transcripts levels of the ETS transcription factor fli-1 are specifically up-regulated in endothelial cells, which overexpress Ets-1, but not in fibroblasts. Analysis of the promoter of the mouse fli-1 gene reveals that the 1-kb region that comprises the transcription starts and part of exon 1 is responsible for the response of the promoter to Ets-1. The -270/-41 fragment contains two known Spi-1-responding Ets binding sites (EBS), which are also necessary for the activation by Ets-1. In contrast to Spi-1, a third EBS is necessary for the full response of this promoter fragment to Ets-1. The rest of the promoter activity has been located in the -986/-505 region, where three active EBSs have been identified. Furthermore, endogenous Fli-1 was found to be bound to its own gene promoter and to be able to promote the transactivation of its gene. These results suggest that Ets-1 activates an auto-regulatory loop of expression of fli-1 in endothelial cells, a mechanism that could have significant implications for the endothelial cell fate.