RESUMEN
Endothelial cell migration and proliferation are essential for the establishment of a hierarchical organization of blood vessels and optimal distribution of blood. However, how these cellular processes are quantitatively coordinated to drive vascular network morphogenesis remains unknown. Here, using the zebrafish vasculature as a model system, we demonstrate that the balanced distribution of endothelial cells, as well as the resulting regularity of vessel calibre, is a result of cell migration from veins towards arteries and cell proliferation in veins. We identify the Wiskott-Aldrich Syndrome protein (WASp) as an important molecular regulator of this process and show that loss of coordinated migration from veins to arteries upon wasb depletion results in aberrant vessel morphology and the formation of persistent arteriovenous shunts. We demonstrate that WASp achieves its function through the coordination of junctional actin assembly and PECAM1 recruitment and provide evidence that this is conserved in humans. Overall, we demonstrate that functional vascular patterning in the zebrafish trunk is established through differential cell migration regulated by junctional actin, and that interruption of differential migration may represent a pathomechanism in vascular malformations.
Asunto(s)
Vasos Sanguíneos/crecimiento & desarrollo , Morfogénesis/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Proteína del Síndrome de Wiskott-Aldrich/genética , Actinas/genética , Animales , Arterias/crecimiento & desarrollo , Arterias/metabolismo , Movimiento Celular/genética , Proliferación Celular/genética , Células Endoteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Uniones Intercelulares/genética , Venas/crecimiento & desarrollo , Venas/metabolismo , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Molecular mechanisms controlling the formation, stabilisation and maintenance of blood vessel connections remain poorly defined. Here, we identify blood flow and the large extracellular protein Svep1 as co-modulators of vessel anastomosis during developmental angiogenesis in zebrafish embryos. Both loss of Svep1 and blood flow reduction contribute to defective anastomosis of intersegmental vessels. The reduced formation and lumenisation of the dorsal longitudinal anastomotic vessel (DLAV) is associated with a compensatory increase in Vegfa/Vegfr pERK signalling, concomittant expansion of apelin-positive tip cells, but reduced expression of klf2a. Experimentally, further increasing Vegfa/Vegfr signalling can rescue the DLAV formation and lumenisation defects, whereas its inhibition dramatically exacerbates the loss of connectivity. Mechanistically, our results suggest that flow and Svep1 co-regulate the stabilisation of vascular connections, in part by modulating the Vegfa/Vegfr signalling pathway.
Asunto(s)
Proteínas de Pez Cebra , Pez Cebra , Anastomosis Quirúrgica , Animales , Morfogénesis , Neovascularización Fisiológica/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Previous studies have shown that Vasohibin 1 (Vash1) is stimulated by VEGFs in endothelial cells and that its overexpression interferes with angiogenesis in vivo Recently, Vash1 was found to mediate tubulin detyrosination, a post-translational modification that is implicated in many cell functions, such as cell division. Here, we used the zebrafish embryo to investigate the cellular and subcellular mechanisms of Vash1 on endothelial microtubules during formation of the trunk vasculature. We show that microtubules within venous-derived secondary sprouts are strongly and selectively detyrosinated in comparison with other endothelial cells, and that this difference is lost upon vash1 knockdown. Vash1 depletion in zebrafish specifically affected secondary sprouting from the posterior cardinal vein, increasing endothelial cell divisions and cell number in the sprouts. We show that altering secondary sprout numbers and structure upon Vash1 depletion leads to defective lymphatic vessel formation and ectopic lymphatic progenitor specification in the zebrafish trunk.
Asunto(s)
Proteínas de Ciclo Celular/genética , Desarrollo Embrionario/genética , Linfangiogénesis/genética , Pez Cebra/embriología , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Secuencia Conservada , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Microtúbulos/metabolismo , Modelos BiológicosRESUMEN
Since its initial characterization, Escherichia coli RNase I has been described as a single-strand specific RNA endonuclease that cleaves its substrate in a largely sequence independent manner. Here, we describe a strong calcium (Ca2+)-dependent activity of RNase I on double-stranded RNA (dsRNA), and a Ca2+-dependent novel hybridase activity, digesting the RNA strand in a DNA:RNA hybrid. Surprisingly, Ca2+ does not affect the activity of RNase I on single stranded RNA (ssRNA), suggesting a specific role for Ca2+ in the modulation of RNase I activity. Mutation of a previously overlooked Ca2+ binding site on RNase I resulted in a gain-of-function enzyme that is highly active on dsRNA and could no longer be stimulated by the metal. In summary, our data imply that native RNase I contains a bound Ca2+, allowing it to target both single- and double-stranded RNAs, thus having a broader substrate specificity than originally proposed for this traditional enzyme. In addition, the finding that the dsRNase activity, and not the ssRNase activity, is associated with the Ca2+-dependency of RNase I may be useful as a tool in applied molecular biology.
Asunto(s)
Calcio/metabolismo , Endorribonucleasas/metabolismo , ARN Bicatenario/metabolismo , Sustitución de Aminoácidos , ADN , Endorribonucleasas/química , Endorribonucleasas/genética , Metales/metabolismo , ARN/metabolismo , Ribonucleasas/metabolismo , Especificidad por SustratoRESUMEN
BACKGROUND: Lymphatic vascular development is regulated by well-characterized signaling and transcriptional pathways. These pathways regulate lymphatic endothelial cell (LEC) migration, motility, polarity, and morphogenesis. Canonical and non-canonical WNT signaling pathways are known to control LEC polarity and development of lymphatic vessels and valves. PKD1, encoding Polycystin-1, is the most commonly mutated gene in polycystic kidney disease but has also been shown to be essential in lymphatic vascular morphogenesis. The mechanism by which Pkd1 acts during lymphangiogenesis remains unclear. RESULTS: Here we find that loss of non-canonical WNT signaling components Wnt5a and Ryk phenocopy lymphatic defects seen in Pkd1 knockout mice. To investigate genetic interaction, we generated Pkd1;Wnt5a double knockout mice. Loss of Wnt5a suppressed phenotypes seen in the lymphatic vasculature of Pkd1-/- mice and Pkd1 deletion suppressed phenotypes observed in Wnt5a-/- mice. Thus, we report mutually suppressive roles for Pkd1 and Wnt5a, with developing lymphatic networks restored to a more wild type state in double mutant mice. This genetic interaction between Pkd1 and the non-canonical WNT signaling pathway ultimately controls LEC polarity and the morphogenesis of developing vessel networks. CONCLUSION: Our work suggests that Pkd1 acts at least in part by regulating non-canonical WNT signaling during the formation of lymphatic vascular networks.
Asunto(s)
Vasos Linfáticos , Enfermedades Renales Poliquísticas , Animales , Vasos Linfáticos/metabolismo , Ratones , Ratones Noqueados , Morfogénesis/genética , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/metabolismo , Proteína Quinasa C , Proteínas Tirosina Quinasas Receptoras/metabolismo , Vía de Señalización Wnt/genética , Proteína Wnt-5a/genética , Proteína Wnt-5a/metabolismoRESUMEN
How developing vascular networks acquire the right balance of arteries, veins and lymphatic vessels to efficiently supply and drain tissues is poorly understood. In zebrafish embryos, the robust and regular 50:50 global balance of intersegmental veins and arteries that form along the trunk prompts the intriguing question of how does the organism keep 'count'? Previous studies have suggested that the ultimate fate of an intersegmental vessel (ISV) is determined by the identity of the approaching secondary sprout emerging from the posterior cardinal vein. Here, we show that the formation of a balanced trunk vasculature involves an early heterogeneity in endothelial cell behaviour and Notch signalling activity in the seemingly identical primary ISVs that is independent of secondary sprouting and flow. We show that Notch signalling mediates the local patterning of ISVs, and an adaptive flow-mediated mechanism subsequently fine-tunes the global balance of arteries and veins along the trunk. We propose that this dual mechanism provides the adaptability required to establish a balanced network of arteries, veins and lymphatic vessels.
Asunto(s)
Tipificación del Cuerpo , Receptores Notch/metabolismo , Pez Cebra/embriología , Animales , Arterias/embriología , Polaridad Celular , Células Endoteliales/fisiología , Heterogeneidad Genética , Vasos Linfáticos/embriología , Flujo Sanguíneo Regional , Transducción de Señal , Venas/embriología , Pez Cebra/sangreRESUMEN
The generation and maintenance of genome edited zebrafish lines is typically labor intensive due to the lack of an easy visual read-out for the modification. To facilitate this process, we have developed a novel method that relies on the inclusion of an artificial intron with a transgenic marker (InTraM) within the knock-in sequence of interest, which upon splicing produces a transcript with a precise and seamless modification. We have demonstrated this technology by replacing the stop codon of the zebrafish fli1a gene with a transcriptional activator KALTA4, using an InTraM that enables red fluorescent protein expression in the heart.
Asunto(s)
Edición Génica/métodos , Técnicas de Sustitución del Gen/métodos , Genes Reporteros , Ensayos Analíticos de Alto Rendimiento/métodos , Animales , Sistemas CRISPR-Cas , Factores de Transcripción/genética , Transgenes , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
BACKGROUND: The interplay between Notch and Vegf signaling regulates angiogenesis in the embryo. Notch signaling limits the responsiveness of endothelial cells to Vegf to control sprouting. Despite the importance of this regulatory relationship, much remains to be understood about extrinsic factors that modulate the pathway. RESULTS: During a forward genetic screen for novel regulators of lymphangiogenesis, we isolated a mutant with reduced lymphatic vessel development. This mutant also exhibited hyperbranching arteries, reminiscent of Notch pathway mutants. Positional cloning identified a missense mutation in the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (cad) gene. Cad is essential for UDP biosynthesis, which is necessary for protein glycosylation and de novo biosynthesis of pyrimidine-based nucleotides. Using a transgenic reporter of Notch activity, we demonstrate that Notch signaling is significantly reduced in cad(hu10125) mutants. In this context, genetic epistasis showed that increased endothelial cell responsiveness to Vegfc/Vegfr3 signaling drives excessive artery branching. CONCLUSIONS: These findings suggest important posttranslational modifications requiring Cad as an unappreciated mechanism that regulates Notch/Vegf signaling during angiogenesis.
Asunto(s)
Aspartato Carbamoiltransferasa/metabolismo , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/metabolismo , Dihidroorotasa/metabolismo , Neovascularización Fisiológica/fisiología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Pez Cebra/embriología , Animales , Aspartato Carbamoiltransferasa/genética , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/genética , Dihidroorotasa/genética , Glicosilación , Receptores Notch/genética , Factor C de Crecimiento Endotelial Vascular/genética , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
In recent years, use of the zebrafish embryo as a model organism to study vascular development in vivo has provided valuable insights into the genetic and cellular events shaping the embryonic vasculature. In this chapter, we aim to present the methods for the measurement of some of the most commonly investigated dynamic parameters in endothelial cells during developmental angiogenesis, namely, migration speed and acceleration, filopodia extension, front-rear polarity, cell cycle progression, membrane deformations, and junctional rearrangements. We also offer suggestions on how to deal with the most common imaging and quantifications challenges faced when acquiring and quantifying endothelial cell dynamic behavior in vivo.We intend this section to serve as an experience-based imaging primer for scientists interested in endothelial cell imaging in the zebrafish embryo.
Asunto(s)
Células Endoteliales/metabolismo , Imagen Molecular , Pez Cebra , Animales , Animales Modificados Genéticamente , Biomarcadores , Ciclo Celular , Membrana Celular/metabolismo , Membrana Celular/patología , Embrión no Mamífero , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Uniones Intercelulares , Imagen Molecular/métodosRESUMEN
Lymphatic vessels arise during development through sprouting of precursor cells from veins, which is regulated by known signaling and transcriptional mechanisms. The ongoing elaboration of vessels to form a network is less well understood. This involves cell polarization, coordinated migration, adhesion, mixing, regression, and shape rearrangements. We identified a zebrafish mutant, lymphatic and cardiac defects 1 (lyc1), with reduced lymphatic vessel development. A mutation in polycystic kidney disease 1a was responsible for the phenotype. PKD1 is the most frequently mutated gene in autosomal dominant polycystic kidney disease (ADPKD). Initial lymphatic precursor sprouting is normal in lyc1 mutants, but ongoing migration fails. Loss of Pkd1 in mice has no effect on precursor sprouting but leads to failed morphogenesis of the subcutaneous lymphatic network. Individual lymphatic endothelial cells display defective polarity, elongation, and adherens junctions. This work identifies a highly selective and unexpected role for Pkd1 in lymphatic vessel morphogenesis during development.