RESUMEN
During angiogenesis, VEGF acts as an attractive cue for endothelial cells (ECs), while Sema3E mediates repulsive cues. Here, we show that the small GTPase RhoJ integrates these opposing signals in directional EC migration. In the GTP-bound state, RhoJ interacts with the cytoplasmic domain of PlexinD1. Upon Sema3E stimulation, RhoJ released from PlexinD1 induces cell contraction. PlexinD1-bound RhoJ further facilitates Sema3E-induced PlexinD1-VEGFR2 association, VEGFR2 transphosphorylation at Y1214, and p38 MAPK activation, leading to reverse EC migration. Upon VEGF stimulation, RhoJ is required for the formation of the holoreceptor complex comprising VEGFR2, PlexinD1, and neuropilin-1, thereby preventing degradation of internalized VEGFR2, prolonging downstream signal transductions via PLCγ, Erk, and Akt, and promoting forward EC migration. After conversion to the GDP-bound state, RhoJ shifts from PlexinD1 to VEGFR2, which then terminates the VEGFR2 signals. RhoJ deficiency in ECs efficiently suppressed aberrant angiogenesis in ischemic retina. These findings suggest that distinct Rho GTPases may act as context-dependent integrators of chemotactic cues in directional cell migration and may serve as candidate therapeutic targets to manipulate cell motility in disease or tissue regeneration.
Asunto(s)
Movimiento Celular , Células Endoteliales/metabolismo , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Animales , Células Endoteliales/citología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Transgénicos , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Unión al GTP rho/genéticaRESUMEN
Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.
Asunto(s)
Actinas , Proteínas de Unión al GTP rap1 , Animales , Ratones , Actinas/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar , Adhesión Celular/fisiología , Endotelio Vascular/metabolismo , Lipopolisacáridos/metabolismo , Pulmón/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
In order to efficiently derive hematopoietic stem cells (HSCs) from pluripotent precursors, it is crucial to understand how mesodermal cells acquire hematopoietic and endothelial identities: two divergent, but closely related, cell fates. Although Npas4 has been recently identified as a conserved master regulator of hemato-vascular development, the molecular mechanisms underlying cell fate divergence between hematopoietic and vascular endothelial cells are still unclear. Here, we show in zebrafish that mesodermal cell differentiation into hematopoietic and vascular endothelial cells is regulated by Junctional adhesion molecule 3b (Jam3b) via two independent signaling pathways. Mutation of jam3b led to a reduction in npas4l expression in the posterior lateral plate mesoderm and defects in both hematopoietic and vascular development. Mechanistically, we show that Jam3b promotes endothelial specification by regulating npas4l expression through repression of the Rap1a-Erk signaling cascade. Jam3b subsequently promotes hematopoietic development, including HSCs, by regulating lrrc15 expression in endothelial precursors through the activation of an integrin-dependent signaling cascade. Our data provide insight into the divergent mechanisms for instructing hematopoietic or vascular fates from mesodermal cells.
Asunto(s)
Sistema Cardiovascular/embriología , Hematopoyesis , Receptores de Superficie Celular/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sistema Cardiovascular/citología , Células Endoteliales/fisiología , Hematopoyesis/fisiología , Células Madre Hematopoyéticas , Sistema de Señalización de MAP Quinasas , Mesodermo/embriología , Receptores de Superficie Celular/genética , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Platelet derived growth factor beta and its receptor, Pdgfrb, play essential roles in the development of vascular mural cells, including pericytes and vascular smooth muscle cells. To determine if this role was conserved in zebrafish, we analyzed pdgfb and pdgfrb mutant lines. Similar to mouse, pdgfb and pdgfrb mutant zebrafish lack brain pericytes and exhibit anatomically selective loss of vascular smooth muscle coverage. Despite these defects, pdgfrb mutant zebrafish did not otherwise exhibit circulatory defects at larval stages. However, beginning at juvenile stages, we observed severe cranial hemorrhage and vessel dilation associated with loss of pericytes and vascular smooth muscle cells in pdgfrb mutants. Similar to mouse, pdgfrb mutant zebrafish also displayed structural defects in the glomerulus, but normal development of hepatic stellate cells. We also noted defective mural cell investment on coronary vessels with concomitant defects in their development. Together, our studies support a conserved requirement for Pdgfrb signaling in mural cells. In addition, these zebrafish mutants provide an important model for definitive investigation of mural cells during early embryonic stages without confounding secondary effects from circulatory defects.
Asunto(s)
Músculo Liso Vascular/metabolismo , Pericitos/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Diferenciación Celular , Vasos Coronarios/metabolismo , Desarrollo Embrionario , Músculo Liso Vascular/embriología , Miocitos del Músculo Liso/metabolismo , Proteínas Proto-Oncogénicas c-sis/metabolismo , Proteínas Proto-Oncogénicas c-sis/fisiología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Transducción de Señal/genética , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases.
Asunto(s)
Proteínas F-Box , Histonas , Proliferación Celular , Células Endoteliales/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Factores de Transcripción/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Mural cells (MCs) are essential for blood vessel stability and function; however, the mechanisms that regulate MC development remain incompletely understood, in particular those involved in MC specification. Here, we investigated the first steps of MC formation in zebrafish using transgenic reporters. Using pdgfrb and abcc9 reporters, we show that the onset of expression of abcc9, a pericyte marker in adult mice and zebrafish, occurs almost coincidentally with an increment in pdgfrb expression in peri-arterial mesenchymal cells, suggesting that these transcriptional changes mark the specification of MC lineage cells from naïve pdgfrblow mesenchymal cells. The emergence of peri-arterial pdgfrbhigh MCs required Notch signaling. We found that pdgfrb-positive cells express notch2 in addition to notch3, and although depletion of notch2 or notch3 failed to block MC emergence, embryos depleted of both notch2 and notch3 lost mesoderm- as well as neural crest-derived pdgfrbhigh MCs. Using reporters that read out Notch signaling and Notch2 receptor cleavage, we show that Notch activation in the mesenchyme precedes specification into pdgfrbhigh MCs. Taken together, these results show that Notch signaling is necessary for peri-arterial MC specification.
Asunto(s)
Arterias/citología , Arterias/embriología , Tipificación del Cuerpo , Mesodermo/embriología , Receptores Notch/metabolismo , Transducción de Señal , Pez Cebra/embriología , Animales , Biomarcadores/metabolismo , Endotelio Vascular/metabolismo , Mesodermo/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Imagen de Lapso de Tiempo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The vascular permeability of the endothelium is finely controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions. In the majority of normal adult tissues, endothelial cells in blood vessels maintain vascular permeability at a relatively low level, while in response to inflammation, they limit vascular barrier function to induce plasma leakage and extravasation of immune cells as a defense mechanism. Thus, the dynamic but also simultaneously tight regulation of vascular permeability by endothelial cells is responsible for maintaining homeostasis and, as such, impairments of its underlying mechanisms result in hyperpermeability, leading to the development and progression of various diseases including coronavirus disease 2019 (COVID-19), a newly emerging infectious disease. Recently, increasing numbers of studies have been unveiling the important role of Rap1, a small guanosine 5'-triphosphatase (GTPase) belonging to the Ras superfamily, in the regulation of vascular permeability. Rap1 enhances VE-cadherin-mediated endothelial cell-cell junctions to potentiate vascular barrier functions via dynamic reorganization of the actin cytoskeleton. Importantly, Rap1 signaling activation reportedly improves vascular barrier function in animal models of various diseases associated with vascular hyperpermeability, suggesting that Rap1 might be an ideal target for drugs intended to prevent vascular barrier dysfunction. Here, we describe recent progress in understanding the mechanisms by which Rap1 potentiates VE-cadherin-mediated endothelial cell-cell adhesions and vascular barrier function. We also discuss how alterations in Rap1 signaling are related to vascular barrier dysfunction in diseases such as acute pulmonary injury and malignancies. In addition, we examine the possibility of Rap1 signaling as a target of drugs for treating diseases associated with vascular hyperpermeability.
Asunto(s)
Antígenos CD/metabolismo , Cadherinas/metabolismo , Permeabilidad Capilar , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Uniones Intercelulares/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Animales , HumanosRESUMEN
The heart is an endocrine organ, as cardiomyocytes (CMs) secrete natriuretic peptide (NP) hormones. Since the discovery of NPs, no other peptide hormones that affect remote organs have been identified from the heart. We identified osteocrin (Ostn) as an osteogenesis/chondrogenesis regulatory hormone secreted from CMs in zebrafish. ostn mutant larvae exhibit impaired membranous and chondral bone formation. The impaired bones were recovered by CM-specific overexpression of OSTN. We analyzed the parasphenoid (ps) as a representative of membranous bones. In the shortened ps of ostn morphants, nuclear Yap1/Wwtr1-dependent transcription was increased, suggesting that Ostn might induce the nuclear export of Yap1/Wwtr1 in osteoblasts. Although OSTN is proposed to bind to NPR3 (clearance receptor for NPs) to enhance the binding of NPs to NPR1 or NPR2, OSTN enhanced C-type NP (CNP)-dependent nuclear export of YAP1/WWTR1 of cultured mouse osteoblasts stimulated with saturable CNP. OSTN might therefore activate unidentified receptors that augment protein kinase G signaling mediated by a CNP-NPR2 signaling axis. These data demonstrate that Ostn secreted from the heart contributes to bone formation as an endocrine hormone.
Asunto(s)
Condrogénesis/genética , Miocitos Cardíacos/metabolismo , Osteogénesis/genética , Cráneo/embriología , Factores de Transcripción/fisiología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Estructuras Animales/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Condrogénesis/efectos de los fármacos , Embrión no Mamífero , Células HEK293 , Corazón/metabolismo , Humanos , Ratones , Organogénesis/efectos de los fármacos , Organogénesis/genética , Osteogénesis/efectos de los fármacos , Hormonas Peptídicas/genética , Hormonas Peptídicas/metabolismo , Hormonas Peptídicas/farmacología , Hormonas Peptídicas/fisiología , Cráneo/efectos de los fármacos , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/farmacologíaRESUMEN
Angiogenesis, the growth of new blood vessels from pre-existing vessels, is critical for cutaneous wound healing. However, it remains elusive how endothelial cells (ECs) and pericytes (PCs) establish new blood vessels during cutaneous angiogenesis. We set up a live-imaging system to analyze cutaneous angiogenesis in adult zebrafish. First, we characterized basic structures of cutaneous vasculature. In normal skin tissues, ECs and PCs remained dormant to maintain quiescent blood vessels, whereas cutaneous injury immediately induced angiogenesis through the vascular endothelial growth factor signaling pathway. Tortuous and disorganized vessel networks formed within a few weeks after the injury and subsequently normalized through vessel regression in a few months. Analyses of the repair process of injured single blood vessels revealed that severed vessels elongated upon injury and anastomosed with each other. Thereafter, repaired vessels and adjacent uninjured vessels became tortuous by increasing the number of ECs. In parallel, PCs divided and migrated to cover the tortuous blood vessels. ECs sprouted from the PC-covered tortuous vessels, suggesting that EC sprouting does not require PC detachment from the vessel wall. Thus, live imaging of cutaneous angiogenesis in adult zebrafish enables us to clarify how ECs and PCs develop new blood vessels during cutaneous angiogenesis.
Asunto(s)
Neovascularización Fisiológica/fisiología , Imagen Óptica/métodos , Fenómenos Fisiológicos de la Piel , Cicatrización de Heridas/fisiología , Envejecimiento/fisiología , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Microscopía Confocal/métodos , Piel/lesiones , Piel/patología , Piel/ultraestructura , Grabación en Video/métodosRESUMEN
Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs.
Asunto(s)
Células Endoteliales/citología , Músculo Liso Vascular/citología , Pericitos/citología , Animales , Animales Modificados Genéticamente , Vasos Sanguíneos/citología , Vasos Sanguíneos/embriología , Microscopía Confocal , Pez CebraRESUMEN
ß-catenin regulates the transcription of genes involved in diverse biological processes, including embryogenesis, tissue homeostasis and regeneration. Endothelial cell (EC)-specific gene-targeting analyses in mice have revealed that ß-catenin is required for vascular development. However, the precise function of ß-catenin-mediated gene regulation in vascular development is not well understood, since ß-catenin regulates not only gene expression but also the formation of cell-cell junctions. To address this question, we have developed a novel transgenic zebrafish line that allows the visualization of ß-catenin transcriptional activity specifically in ECs and discovered that ß-catenin-dependent transcription is central to the bone morphogenetic protein (Bmp)-mediated formation of venous vessels. During caudal vein (CV) formation, Bmp induces the expression of aggf1, a putative causative gene for Klippel-Trenaunay syndrome, which is characterized by venous malformation and hypertrophy of bones and soft tissues. Subsequently, Aggf1 potentiates ß-catenin transcriptional activity by acting as a transcriptional co-factor, suggesting that Bmp evokes ß-catenin-mediated gene expression through Aggf1 expression. Bmp-mediated activation of ß-catenin induces the expression of Nr2f2 (also known as Coup-TFII), a member of the nuclear receptor superfamily, to promote the differentiation of venous ECs, thereby contributing to CV formation. Furthermore, ß-catenin stimulated by Bmp promotes the survival of venous ECs, but not that of arterial ECs. Collectively, these results indicate that Bmp-induced activation of ß-catenin through Aggf1 regulates CV development by promoting the Nr2f2-dependent differentiation of venous ECs and their survival. This study demonstrates, for the first time, a crucial role of ß-catenin-mediated gene expression in the development of venous vessels.
Asunto(s)
Células Endoteliales/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Venas/embriología , beta Catenina/metabolismo , Proteínas Angiogénicas/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Morfogenéticas Óseas/metabolismo , Factor de Transcripción COUP II/metabolismo , ADN Complementario/genética , Células Endoteliales/ultraestructura , Células HEK293 , Humanos , Etiquetado Corte-Fin in Situ , Luciferasas , Proteínas Luminiscentes , Microscopía Fluorescente , Morfolinos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Venas/citología , Pez Cebra , Proteínas de Pez Cebra/metabolismo , Proteína Fluorescente RojaRESUMEN
Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. Via its receptor, guanylyl cyclase-A (GC-A), ANP maintains cardiovascular homeostasis by exerting diuretic, natriuretic, and hypotensive effects mediated, in part, by endothelial cells. Both in vivo and in vitro, ANP enhances endothelial barrier function by reducing RhoA activity and reorganizing the actin cytoskeleton. We established mouse endothelial cells that stably express GC-A and used them to analyze the molecular mechanisms responsible for actin reorganization. Stimulation by ANP resulted in phosphorylation of myosin light chain (MLC) and promotion of cell spreading. p21-activated kinase 4 (PAK4) and cerebral cavernous malformations 2 (CCM2), a scaffold protein involved in a cerebrovascular disease, were required for the phosphorylation of MLC and promotion of cell spreading by ANP. Finally, in addition to the GC domain, the kinase homology domain of GC-A was also required for ANP/GC-A signaling. Our results indicate that CCM2 and PAK4 are important downstream mediators of ANP/GC-A signaling involved in cell spreading, an important initial step in the enhancement of endothelial barrier function.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Factor Natriurético Atrial/metabolismo , Proteínas Portadoras/agonistas , Endotelio Vascular/metabolismo , Receptores del Factor Natriurético Atrial/agonistas , Transducción de Señal , Quinasas p21 Activadas/metabolismo , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Bovinos , Movimiento Celular , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Activación Enzimática , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Mutación , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Interferencia de ARN , Receptores del Factor Natriurético Atrial/química , Receptores del Factor Natriurético Atrial/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/química , Quinasas p21 Activadas/genéticaRESUMEN
Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA.
Asunto(s)
Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Membrana Celular/enzimología , Movimiento Celular , Quimiocina CXCL12/fisiología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Células Endoteliales/fisiología , Factores de Intercambio de Guanina Nucleótido/química , Células HEK293 , Humanos , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
Blood vessels and neurons grow often side by side. However, the molecular and cellular mechanisms underlying their parallel development remain unclear. Here, we report that a subpopulation of secondary motoneurons extends axons ventrally outside of the neural tubes and rostrocaudally as a fascicle beneath the dorsal aorta (DA) in zebrafish. We tried to clarify the mechanism by which these motoneuron axons grow beneath the DA and found that Vegfc in the DA and Vegfr3 in the motoneurons were essential for the axon growth. Forced expression of either Vegfc in arteries or Vegfr3 in motoneurons resulted in enhanced axon growth of motoneurons over the DA. Both vegfr3 morphants and vegfc morphants lost the alignment of motoneuron axons with DA. In addition, forced expression of two mutant forms of Vegfr3 in motoneurons, potentially trapping endogenous Vegfc, resulted in failure of growth of motoneuron axons beneath the DA. Finally, a vegfr3 mutant fish lacked the motoneuron axons beneath the DA. Collectively, Vegfc from the preformed DA guides the axon growth of secondary motoneurons.
Asunto(s)
Aorta/citología , Aorta/metabolismo , Axones/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Factor C de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Transducción de Señal/genética , Transducción de Señal/fisiología , Factor C de Crecimiento Endotelial Vascular/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.
Asunto(s)
Ciclo Celular/genética , Endotelio Vascular/fisiología , Neovascularización Fisiológica/genética , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/genética , Ciclo Celular/fisiología , División Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Endotelio Vascular/citología , Técnicas de Silenciamiento del Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Morfolinos/genética , Pez Cebra/genética , Proteínas de Pez Cebra/biosíntesisRESUMEN
Development requires cell proliferation, migration, differentiation, apoptosis, and many kinds of cell responses. Cells prepare intracellular conditions to respond to extracellular cues from neighboring cells. We have studied the development of the cardiovascular system (CVS) by visualizing morphology and signaling simultaneously using zebrafish, which express probes for both. Endodermal sheet is required for the bilateral cardiac precursor cell (CPC) migration toward the midline. Endothelial cells (ECs) proliferate specifically in the certain regions of blood vessels. Bone morphogenetic proteins (BMP) induce the remodeling of the caudal vein plexus (CVP) to form the caudal vein (CV). Our findings point to the pre-existing neighboring cells as the cells exhibiting certain responses during the development of CVS. In this review, we introduce recent results of our research on angiogenesis and cardiogenesis by spotlighting the mechanism by which ECs and CPCs are regulated by the cells next to themselves. In addition, we discuss the unanswered questions that should be clarified in the future in the field of CVS development.
Asunto(s)
Sistema Cardiovascular/embriología , Pez Cebra/embriología , Animales , Movimiento Celular , Proliferación Celular , Activación TranscripcionalRESUMEN
G protein-coupled receptors (GPCRs) linked to both members of the Gα12 family of heterotrimeric G proteins α subunits, Gα12 and Gα13, regulate the activation of Rho GTPases, thereby contributing to many key biological processes. Multiple Rho GEFs have been proposed to link Gα12/13 GPCRs to Rho activation, including PDZ-RhoGEF (PRG), leukemia-associated Rho GEF (LARG), p115-RhoGEF (p115), lymphoid blast crisis (Lbc), and Dbl. PRG, LARG, and p115 share the presence of a regulator of G protein signaling homology (RGS) domain. There is limited information on the biological roles of this RGS-containing family of RhoGEFs in vivo. p115-deficient mice are viable with some defects in the immune system and gastrointestinal motor dysfunctions, whereas in an initial study we showed that mice deficient for Larg are viable and resistant to salt-induced hypertension. Here, we generated knock-out mice for Prg and observed that these mice do not display any overt phenotype. However, deficiency in Prg and Larg leads to complex developmental defects and early embryonic lethality. Signaling from Gα11/q-linked GPCRs to Rho was not impaired in mouse embryonic fibroblasts defective in all three RGS-containing RhoGEFs. However, a combined lack of Prg, Larg, and p115 expression abolished signaling through Gα12/13 to Rho and thrombin-induced cell proliferation, directional migration, and nuclear signaling through JNK and p38. These findings provide evidence of an essential role for the RGS-containing RhoGEF family in signaling to Rho by Gα12/13-coupled GPCRs, which may likely play a critical role during embryonic development.
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Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Receptores de Trombina/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Línea Celular , Movimiento Celular/fisiología , Proliferación Celular , Fibroblastos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/genética , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Ratones , Ratones Noqueados , Receptores del Ácido Lisofosfatídico/genética , Receptores de Trombina/genética , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/genéticaRESUMEN
Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown. Herein, we show that ECs regulate alveologenesis by constructing basement membranes (BMs) acting as a scaffold for myofibroblasts to induce septa formation through activating mechanical signaling. Rap1, a small GTPase of the Ras superfamily, is known to stimulate integrin-mediated cell adhesions. EC-specific Rap1-deficient (Rap1iECKO) mice exhibit impaired septa formation and hypo-alveolarization due to the decreased mechanical signaling in myofibroblasts. In Rap1iECKO mice, ECs fail to stimulate integrin ß1 to recruit Collagen type IV (Col-4) into BMs required for myofibroblast-mediated septa formation. Consistently, EC-specific integrin ß1-deficient mice show hypo-alveolarization, defective mechanical signaling in myofibroblasts, and disorganized BMs. These data demonstrate that alveolar ECs promote integrin ß1-mediated Col-4 recruitment in a Rap1-dependent manner, thereby constructing BMs acting as a scaffold for myofibroblasts to induce mechanical signal-mediated alveologenesis. Thus, this study unveils a mechanism of organ morphogenesis mediated by ECs through intrinsic functions.
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Células Endoteliales , Miofibroblastos , Animales , Ratones , Membrana Basal , Integrina beta1/genética , MorfogénesisRESUMEN
Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.
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Encéfalo , Imagenología Tridimensional , Microscopía Fluorescente , Animales , Ratones , Encéfalo/diagnóstico por imagen , Humanos , Microscopía Fluorescente/métodos , Microscopía Fluorescente/instrumentación , Imagenología Tridimensional/métodos , Línea Celular TumoralRESUMEN
Angiogenesis is a dynamic morphogenetic process that refers to the growth of new blood vessels from the pre-existing vessels and is critical for tissue repair during wound healing. In adult normal tissues, quiescent endothelial cells and pericytes maintain vascular integrity, whereas angiogenesis is immediately induced upon tissue injury, thereby forming neovascular networks to maintain homeostasis. However, impaired angiogenesis results in development of chronic and non-healing wounds in various diseases such as diabetes and peripheral artery diseases. Zebrafish are a vertebrate model organism widely used for studying many medical and life science fields. Indeed, the molecular and cellular mechanisms underlying regulation of wound angiogenesis have recently been studied by performing fluorescence-based live-imaging of adult zebrafish. In this review, we describe how endothelial cells and pericytes establish neovascular networks during wound angiogenesis and also introduce a novel role of blood flow-driven intraluminal pressure in regulating angiogenesis during wound healing.