RESUMEN
Vessel formation has been extensively studied at the tissue level, but the difficulty in imaging the endothelium with cellular resolution has hampered study of the morphogenesis and behavior of endothelial cells (ECs) in vivo. We are using endothelial-specific transgenes and high-resolution imaging to examine single ECs in zebrafish. By generating mosaics with transgenes that simultaneously mark endothelial nuclei and membranes we are able to definitively identify and study the morphology and behavior of individual ECs during vessel sprouting and lumen formation. Using these methods, we show that developing trunk vessels are composed of ECs of varying morphology, and that single-cell analysis can be used to quantitate alterations in morphology and dynamics in ECs that are defective in proper guidance and patterning. Finally, we use single-cell analysis of intersegmental vessels undergoing lumen formation to demonstrate the coexistence of seamless transcellular lumens and single or multicellular enclosed lumens with autocellular or intercellular junctions, suggesting that heterogeneous mechanisms contribute to vascular lumen formation in vivo. The tools that we have developed for single EC analysis should facilitate further rigorous qualitative and quantitative analysis of EC morphology and behavior in vivo.
Asunto(s)
Endotelio/citología , Endotelio/embriología , Morfogénesis , Análisis de la Célula Individual/métodos , Pez Cebra/embriología , Animales , Polaridad Celular , Embrión no Mamífero/citología , Células Endoteliales/citología , Endotelio/irrigación sanguínea , Imagenología Tridimensional , Uniones Intercelulares , Espacio Intracelular/metabolismo , Fusión de Membrana , Neovascularización Fisiológica , Reproducibilidad de los Resultados , Torso/irrigación sanguínea , Torso/embriología , Vacuolas/metabolismoRESUMEN
The cilium is a signaling platform of the vertebrate cell. It has a critical role in polycystic kidney disease and nephronophthisis. Cilia have been detected on endothelial cells, but the function of these organelles in the vasculature remains incompletely defined. In this study, using genetic and chemical genetic tools in the model organism zebrafish, we reveal an essential role of cilia in developmental vascular integrity. Embryos expressing mutant intraflagellar transport genes, which are essential and specific for cilia biogenesis, displayed increased risk of developmental intracranial hemorrhage, whereas the morphology of the vasculature remained normal. Moreover, cilia were present on endothelial cells in the developing zebrafish vasculature. We further show that the involvement of cilia in vascular integrity is endothelial autonomous, because endothelial-specific re-expression of intraflagellar transport genes in respective mutants rescued the intracranial hemorrhage phenotype. Finally, whereas inhibition of Hedgehog signaling increased the risk of intracranial hemorrhage in ciliary mutants, activation of the pathway rescued this phenotype. In contrast, embryos expressing an inactivating mutation in pkd2, one of two autosomal dominant cystic kidney disease genes, did not show increased risk of developmental intracranial hemorrhage. These results suggest that Hedgehog signaling is a major mechanism for this novel role of endothelial cilia in establishing vascular integrity.
Asunto(s)
Cilios/fisiología , Endotelio Vascular/fisiología , Proteínas Hedgehog/metabolismo , Hemorragias Intracraneales/etiología , Animales , Células Endoteliales/citología , Mecanotransducción Celular , Canales Catiónicos TRPP/fisiología , Pez CebraRESUMEN
Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Desarrollo Embrionario/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Morfogénesis/fisiología , Transducción de Señal/fisiología , Pez Cebra/embriología , Animales , Secuencia de Bases , Proteínas Activadoras de GTPasa/genética , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos de 4 o más Anillos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Cadenas Ligeras de Miosina/metabolismo , Fosforilación , Filogenia , Análisis de Secuencia de ADN , Imagen de Lapso de Tiempo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Cell adhesion and motility is of fundamental importance during development, normal physiology and pathologic conditions such as tumor metastasis. Focal adhesion proteins and their dynamic interactions play a critical role in the regulation of directed cell migration upon exposure to extracellular guidance cues. Using a combination of pharmacological inhibitors, knockout and knockdown cells and mutant protein expression, we recently reported that following adhesion and growth factor stimulation the dynamic interaction between paxillin and PKL(GIT2) is regulated by Src/FAK-dependent phosphorylation of PKL and that this interaction is necessary for the coordination of Rho family GTPase signaling controlling front-rear cell polarity and thus directional migration. Herein, we discuss the implications of these observations.
Asunto(s)
Movimiento Celular , Polaridad Celular , Proteínas Activadoras de GTPasa/metabolismo , Paxillin/metabolismo , Animales , Adhesión Celular , Matriz Extracelular/metabolismo , Humanos , Modelos Biológicos , Fosforilación , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Directed cell migration requires the coordination of growth factor and cell adhesion signaling and is of fundamental importance during embryonic development, wound repair, and pathological conditions such as tumor metastasis. Herein, we demonstrate that the ArfGAP, paxillin-kinase-linker (PKL/GIT2), is tyrosine phosphorylated in response to platelet-derived growth factor (PDGF) stimulation, in an adhesion dependent manner and is necessary for directed cell migration. Using a combination of pharmacological inhibitors, knockout cells and kinase mutants, FAK, and Src family kinases were shown to mediate PDGF-dependent PKL tyrosine phosphorylation. In fibroblasts, expression of a PKL mutant lacking the principal tyrosine phosphorylation sites resulted in loss of wound-induced cell polarization as well as directional migration. PKL phosphorylation was necessary for PDGF-stimulated PKL binding to the focal adhesion protein paxillin and expression of paxillin or PKL mutants defective in their respective binding motifs recapitulated the polarization defects. RNA interference or expression of phosphorylation mutants of PKL resulted in disregulation of PDGF-stimulated Rac1 and PAK activities, reduction of Cdc42 and Erk signaling, as well as mislocalization of betaPIX. Together these studies position PKL as an integral component of growth factor and cell adhesion cross-talk signaling, controlling the development of front-rear cell polarity and directional cell migration.