RESUMEN
Transcription factors (TFs) play a crucial role in regulating the dynamic and precise patterns of gene expression required for the initial specification of endothelial cells (ECs), and during endothelial growth and differentiation. While sharing many core features, ECs can be highly heterogeneous. Differential gene expression between ECs is essential to pattern the hierarchical vascular network into arteries, veins and capillaries, to drive angiogenic growth of new vessels, and to direct specialization in response to local signals. Unlike many other cell types, ECs have no single master regulator, instead relying on differing combinations of a necessarily limited repertoire of TFs to achieve tight spatial and temporal activation and repression of gene expression. Here, we will discuss the cohort of TFs known to be involved in directing gene expression during different stages of mammalian vasculogenesis and angiogenesis, with a primary focus on development.
Asunto(s)
Células Endoteliales , Factores de Transcripción , Animales , Humanos , Factores de Transcripción/metabolismo , Células Endoteliales/metabolismo , Angiogénesis , Neovascularización Fisiológica/genética , Arterias , Mamíferos/metabolismoRESUMEN
Developmentally, the great vessels of the heart originate from the pharyngeal arch arteries (PAAs). During PAA vasculogenesis, PAA precursors undergo sequential cell fate decisions that are accompanied by proliferative expansion. However, how these two processes are synchronized remains poorly understood. Here, we find that the zebrafish chemokine receptor Cxcr4a is expressed in PAA precursors, and genetic ablation of either cxcr4a or the ligand gene cxcl12b causes PAA stenosis. Cxcr4a is required for the activation of the downstream PI3K/AKT cascade, which promotes not only PAA angioblast proliferation, but also differentiation. AKT has a well-known role in accelerating cell-cycle progression through the activation of cyclin-dependent kinases. Despite this, we demonstrate that AKT phosphorylates Etv2 and Scl, the key regulators of angioblast commitment, on conserved serine residues, thereby protecting them from ubiquitin-mediated proteasomal degradation. Altogether, our study reveals a central role for chemokine signaling in PAA vasculogenesis through orchestrating angioblast proliferation and differentiation.
Asunto(s)
Región Branquial , Pez Cebra , Animales , Pez Cebra/genética , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Arterias , Quimiocinas , División CelularRESUMEN
Chronic kidney disease (CKD) and end stage renal disease (ESRD) are increasingly frequent and devastating conditions that have driven a surge in the need for kidney transplantation. A stark shortage of organs has fueled interest in generating viable replacement tissues ex vivo for transplantation. One promising approach has been self-organizing organoids, which mimic developmental processes and yield multicellular, organ-specific tissues. However, a recognized roadblock to this approach is that many organoid cell types fail to acquire full maturity and function. Here, we comprehensively assess the vasculature in two distinct kidney organoid models as well as in explanted embryonic kidneys. Using a variety of methods, we show that while organoids can develop a wide range of kidney cell types, as previously shown, endothelial cells (ECs) initially arise but then rapidly regress over time in culture. Vasculature of cultured embryonic kidneys exhibit similar regression. By contrast, engraftment of kidney organoids under the kidney capsule results in the formation of a stable, perfused vasculature that integrates into the organoid. This work demonstrates that kidney organoids offer a promising model system to define the complexities of vascular-nephron interactions, but the establishment and maintenance of a vascular network present unique challenges when grown ex vivo.
Asunto(s)
Endotelio Vascular/embriología , Riñón/irrigación sanguínea , Riñón/embriología , Organogénesis , Organoides/embriología , Animales , Células Cultivadas , Células Endoteliales , Endotelio Vascular/citología , Femenino , Humanos , Riñón/citología , Masculino , Ratones , Organoides/trasplante , RNA-Seq , Técnicas de Cultivo de TejidosRESUMEN
Vascular structures in the developing brain are thought to form via angiogenesis from preformed blood vessels in the cephalic mesenchyme. Immunohistochemical studies of developing mouse brain from E10.5 to E13.5 revealed the presence of avascular blood islands of primitive erythroid cells expressing hemangioblast markers (Flk1, Tal1/Scl1, platelet endothelial cell adhesion molecule 1, vascular endothelial-cadherin, and CD34) and an endothelial marker recognized by Griffonia simplicifolia isolectin B4 (IB4) in the cephalic mesenchyme. These cells formed a perineural vascular plexus from which angiogenic sprouts originated and penetrated the neuroepithelium. In addition, avascular isolated cells expressing primitive erythroid, hemangioblast and endothelial makers were visible in the neuroepithelium where they generated vasculogenic and hemogenic foci. From E10.5 to E13.5, these vasculogenic foci were a source of new blood vessel formation in the developing brain. In vitro, cultured E13.5 brain endothelial cells contained hemogenic endothelial cells capable of generating erythroid cells. Similar cells were present in primary cultures of dissociated cells from E10.5 embryonic head. Our results provide new evidence that the brain vasculature, like that of the yolk sac and the eye choriocapillaris and hyaloid vascular systems, develops at least in part via hemovasculogenesis, a process in which vasculogenesis and hematopoiesis occur simultaneously.
Asunto(s)
Encéfalo/irrigación sanguínea , Encéfalo/embriología , Endotelio Vascular/embriología , Animales , Encéfalo/citología , Endotelio Vascular/citología , Femenino , Ratones , Morfogénesis/fisiología , Embarazo , Saco Vitelino/irrigación sanguínea , Saco Vitelino/citología , Saco Vitelino/embriologíaRESUMEN
The development of a vascular network is essential to nourish tissues and sustain organ function throughout life. Endothelial cells (ECs) are the building blocks of blood vessels, yet our understanding of EC specification remains incomplete. Zebrafish cloche/npas4l mutants have been used broadly as an avascular model, but little is known about the molecular mechanisms of action of the Npas4l transcription factor. Here, to identify its direct and indirect target genes, we have combined complementary genome-wide approaches, including transcriptome analyses and chromatin immunoprecipitation. The cross-analysis of these datasets indicates that Npas4l functions as a master regulator by directly inducing a group of transcription factor genes that are crucial for hematoendothelial specification, such as etv2, tal1 and lmo2 We also identified new targets of Npas4l and investigated the function of a subset of them using the CRISPR/Cas9 technology. Phenotypic characterization of tspan18b mutants reveals a novel player in developmental angiogenesis, confirming the reliability of the datasets generated. Collectively, these data represent a useful resource for future studies aimed to better understand EC fate determination and vascular development.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Endotelio Vascular/embriología , Regulación del Desarrollo de la Expresión Génica , Neovascularización Fisiológica/genética , Proteínas de Pez Cebra/fisiología , Pez Cebra , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión/genética , Diferenciación Celular/genética , Mapeo Cromosómico/métodos , Conjuntos de Datos como Asunto , Embrión no Mamífero , Células Endoteliales/fisiología , Endotelio Vascular/metabolismo , Genómica/métodos , Proteínas con Dominio LIM/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Factores de Transcripción/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Lymphatic vessels are known to be derived from veins; however, recent lineage-tracing experiments propose that specific lymphatic networks may originate from both venous and non-venous sources. Despite this, direct evidence of a non-venous lymphatic progenitor is missing. Here, we show that the zebrafish facial lymphatic network is derived from three distinct progenitor populations that add sequentially to the developing facial lymphatic through a relay-like mechanism. We show that while two facial lymphatic progenitor populations are venous in origin, the third population, termed the ventral aorta lymphangioblast (VA-L), does not sprout from a vessel; instead, it arises from a migratory angioblast cell near the ventral aorta that initially lacks both venous and lymphatic markers, and contributes to the facial lymphatics and the hypobranchial artery. We propose that sequential addition of venous and non-venous progenitors allows the facial lymphatics to form in an area that is relatively devoid of veins. Overall, this study provides conclusive, live imaging-based evidence of a non-venous lymphatic progenitor and demonstrates that the origin and development of lymphatic vessels is context-dependent.
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Vasos Linfáticos/fisiología , Células Madre/fisiología , Venas/fisiología , Pez Cebra/fisiología , Animales , Movimiento Celular/fisiología , Células Endoteliales/fisiologíaRESUMEN
PROBLEM: The role of extracellular signal-regulated kinase (ERK)1/2-mediated angiogenesis during endometriotic nidation is unknown. We posit that ERK1/2-induced angioblast differentiation and proliferation promotes ectopic endometrial angiogenesis. METHODS OF STUDY: Human eutopic and ectopic endometria were immunostained for total- (T-) or phosphorylated- (P-) ERK1/2 or double-immunostained for P-ERK1/2-CD34 and PCNA-CD34. Estradiol (E2 ), cytokines, normal peritoneal fluid (NPF) or endometriotic peritoneal fluid (EPF) ±PD98059, an ERK1/2 inhibitor, treaded primary human endometrial endothelial cells (HEECs) were evaluated by T-/P-ERK1/2 immunoblotting, MTT viability and tube formation assays. RESULTS: HEECs exhibited higher endothelial P-ERK1/2 immunoreactivity in ectopic vs eutopic endometria. Double-immunostained ectopic endometria displayed abundant CD34-positive angioblasts exhibiting strong P-ERK1/2 and PCNA immunoreactivity. EPF and vascular growth factor (VEGF)-A significantly increased HEEC proliferation and P-ERK1/2 levels. PD98059 reduced basal, EPF, and VEGF-induced HEEC proliferation and promoted vascular stabilization following tube formation. CONCLUSION: Enhanced ERK1/2 activity in angioblasts by such peritoneal factors as VEGF, E2 induces proliferation to trigger ectopic endometrial angiogenesis.
Asunto(s)
Coristoma/metabolismo , Endometriosis/metabolismo , Endometrio/patología , Células Endoteliales/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neovascularización Patológica , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Humanos , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Angioblasts of the developing vascular system require many signaling inputs to initiate their migration, proliferation and differentiation into endothelial cells. What is less studied is which intrinsic cell factors interpret these extrinsic signals. Here, we show the Lim homeodomain transcription factor islet2a (isl2a) is expressed in the lateral posterior mesoderm prior to angioblast migration. isl2a deficient angioblasts show disorganized migration to the midline to form axial vessels and fail to spread around the tailbud of the embryo. Isl2a morphants have fewer vein cells and decreased vein marker expression. We demonstrate that isl2a is required cell autonomously in angioblasts to promote their incorporation into the vein, and is permissive for vein identity. Knockout of isl2a results in decreased migration and proliferation of angioblasts during intersegmental artery growth. Since Notch signaling controls both artery-vein identity and tip-stalk cell formation, we explored the interaction of isl2a and Notch. We find that isl2a expression is negatively regulated by Notch activity, and that isl2a positively regulates flt4, a VEGF-C receptor repressed by Notch during angiogenesis. Thus Isl2a may act as an intermediate between Notch signaling and genetic programs controlling angioblast number and migration, placing it as a novel transcriptional regulator of early angiogenesis.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas con Homeodominio LIM/fisiología , Neovascularización Fisiológica/fisiología , Factores de Transcripción/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Arterias/embriología , Movimiento Celular , Técnicas de Inactivación de Genes , Proteínas con Homeodominio LIM/deficiencia , Proteínas con Homeodominio LIM/genética , Mesodermo , Morfolinos/genética , Morfolinos/toxicidad , Neovascularización Patológica/genética , Neovascularización Patológica/patología , ARN Mensajero/genética , Receptores Notch/fisiología , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Transcripción Genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/fisiología , Venas/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Formation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. Although it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels and the molecular mechanisms regulating their formation are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas and, as such, represents an ideal model in which to investigate the early steps of organ-specific vessel formation. Here, we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the posterior cardinal vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein forms and expands ventrally through a Bone Morphogenetic Protein-dependent step of collective migration. Concomitantly, a Vascular Endothelial Growth Factor-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers, is observed, which culminates with the generation of the supraintestinal artery. Together, our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late-forming, tissue-specific vascular beds.
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Desarrollo Embrionario , Especificidad de Órganos , Venas/citología , Venas/embriología , Pez Cebra/embriología , Animales , Arterias/citología , Movimiento Celular , Sistema Digestivo/irrigación sanguínea , Células Endoteliales/citología , Hígado/irrigación sanguínea , Receptores Notch/metabolismo , Vasos Retinianos/metabolismoRESUMEN
A key step in the de novo formation of the embryonic vasculature is the migration of endothelial precursors, the angioblasts, to the position of the future vessels. To form the first axial vessels, angioblasts migrate towards the midline and coalesce underneath the notochord. Vascular endothelial growth factor has been proposed to serve as a chemoattractant for the angioblasts and to regulate this medial migration. Here we challenge this model and instead demonstrate that angioblasts rely on their intrinsic expression of Apelin receptors (Aplr, APJ) for their migration to the midline. We further show that during this angioblast migration Apelin receptor signaling is mainly triggered by the recently discovered ligand Elabela (Ela). As neither of the ligands Ela or Apelin (Apln) nor their receptors have previously been implicated in regulating angioblast migration, we hereby provide a novel mechanism for regulating vasculogenesis, with direct relevance to physiological and pathological angiogenesis.
Asunto(s)
Movimiento Celular/fisiología , Quimiocinas/metabolismo , Células Endoteliales/citología , Células Progenitoras Endoteliales/fisiología , Modelos Biológicos , Neovascularización Fisiológica/fisiología , Proteínas de Pez Cebra/metabolismo , Animales , Clonación Molecular , Cartilla de ADN/genética , Células Progenitoras Endoteliales/metabolismo , Humanos , Hibridación in Situ , Mutagénesis , Pez CebraRESUMEN
Corneal avascularity is important for optical clarity and normal vision. However, the molecular mechanisms that prevent angioblast migration and vascularization of the developing cornea are not clear. Previously we showed that periocular angioblasts and forming ocular blood vessels avoid the presumptive cornea despite dynamic ingression of neural crest cells. In the current study, we investigate the role of Semaphorin3A (Sema3A), a cell guidance chemorepellent, on angioblast migration and corneal avascularity during development. We show that Sema3A, Vegf, and Nrp1 are expressed in the anterior eye during cornea development. Sema3A mRNA transcripts are expressed at significantly higher levels than Vegf in the lens that is positioned adjacent to the presumptive cornea. Blockade of Sema3A signaling via lens removal or injection of a synthetic Sema3A inhibitor causes ectopic migration of angioblasts into the cornea and results in its subsequent vascularization. In addition, using bead implantation, we demonstrate that exogenous Sema3A protein inhibits Vegf-induced vascularization of the cornea. In agreement with these findings, loss of Sema/Nrp1 signaling in Nrp1(Sema-) mutant mice results in ectopic angioblasts and vascularization of the embryonic mouse corneas. Altogether, our results reveal Sema3A signaling as an important cue during the establishment of corneal avascularity in both chick and mouse embryos. Our study introduces cornea development as a new model for studying the mechanisms involved in vascular patterning during embryogenesis and it also provides new insights into therapeutic potential for Sema3A in neovascular diseases.
Asunto(s)
Córnea/irrigación sanguínea , Cristalino/irrigación sanguínea , Neuropilina-1/genética , Semaforina-3A/fisiología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Animales Modificados Genéticamente , Movimiento Celular , Células Cultivadas , Embrión de Pollo , Córnea/embriología , Células Endoteliales , Cristalino/embriología , Ratones , Neovascularización Fisiológica , Neuropilina-1/biosíntesis , Codorniz/embriología , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/genética , Semaforina-3A/antagonistas & inhibidores , Semaforina-3A/genética , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
etv2 is an endothelial-specific ETS transcription factor that is essential for vascular differentiation and morphogenesis in vertebrates. While recent data suggest that Etv2 is dynamically regulated during vascular development, little is known about the mechanisms involved in this process. Here, we find that etv2 transcript and protein expression are highly dynamic during zebrafish vascular development, with both apparent during early somitogenesis and subsequently down-regulated as development proceeds. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis stages, but not later, caused severe vascular defects, suggesting a specific role in early commitment of lateral mesoderm to the endothelial linage. Accordingly, Etv2-overexpressing cells showed an enhanced ability to commit to endothelial lineages in mosaic embryos. We further find that the etv2 3' untranslated region (UTR) is capable of repressing an endothelial autonomous transgene and contains binding sites for members of the let-7 family of microRNAs. Ectopic expression of let-7a could repress the etv2 3'UTR in sensor assays and was also able to block endogenous Etv2 protein expression, leading to concomitant reduction of endothelial genes. Finally, we observed that Etv2 protein levels persisted in maternal-zygotic dicer1 mutant embryos, suggesting that microRNAs contribute to its repression during vascular development. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is then down-regulated, in part through post-transcriptional repression by microRNAs, to allow normal vascular development.
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Transcripción Genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Regiones no Traducidas 3' , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The formation and lumenization of blood vessels has been studied in some detail, but there is little understanding of the morphogenetic mechanisms by which endothelial cells (ECs) forming large caliber vessels aggregate, align themselves and finally form a lumen that can support blood flow. Here, we focus on the development of the zebrafish common cardinal veins (CCVs), which collect all the blood from the embryo and transport it back to the heart. We show that the angioblasts that eventually form the definitive CCVs become specified as a separate population distinct from the angioblasts that form the lateral dorsal aortae. The subsequent development of the CCVs represents a novel mechanism of vessel formation, during which the ECs delaminate and align along the inner surface of an existing luminal space. Thereby, the CCVs are initially established as open-ended endothelial tubes, which extend as single EC sheets along the flow routes of the circulating blood and eventually enclose the entire lumen in a process that we term 'lumen ensheathment'. Furthermore, we found that the initial delamination of the ECs as well as the directional migration within the EC sheet depend on Cadherin 5 function. By contrast, EC proliferation within the growing CCV is controlled by Vascular endothelial growth factor C, which is provided by circulating erythrocytes. Our findings not only identify a novel mechanism of vascular lumen formation, but also suggest a new form of developmental crosstalk between hematopoietic and endothelial cell lineages.