RESUMEN
Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.
Asunto(s)
Mutación con Pérdida de Función , Proteínas de Transporte Vesicular , Animales , Ratones , Humanos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Células Endoteliales/metabolismo , Secreción de Insulina , Colesterol , Mamíferos/metabolismoRESUMEN
Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.
RESUMEN
Vascular endothelial growth factor receptor 3 (Vegfr3) has been widely used as a marker for lymphatic and vascular endothelial cells during mouse embryonic development and in adult mouse, making it valuable for studying angiogenesis and lymphangiogenesis under normal and pathological conditions. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a membrane-localized fluorescent reporter protein, Gap43-Venus, under the control of the Vegfr3 regulatory sequence. Vegfr3-Gap43-Venus BAC Tg recapitulated endogenous Vegfr3 expression in vascular and lymphatic endothelial cells during embryonic development and tumor development. Thus, this Tg mouse line contributes a valuable model to study angiogenesis and lymphangiogenesis in physiological and pathological contexts.
Asunto(s)
Proteínas Bacterianas/metabolismo , Células Endoteliales/metabolismo , Proteína GAP-43/metabolismo , Proteínas Luminiscentes/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Proteínas Bacterianas/genética , Vasos Sanguíneos/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Embrión de Mamíferos/irrigación sanguínea , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Proteína GAP-43/genética , Expresión Génica , Proteínas Luminiscentes/genética , Vasos Linfáticos/citología , Vasos Linfáticos/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos , Microscopía Confocal , Neoplasias Experimentales/irrigación sanguínea , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Fibroblast growth factor 5 (Fgf5) has been widely used as a marker for the epiblast in the postimplantation embryo and epiblast stem cells (mEpiSCs) in the mouse, making it valuable for study of differentiation of various tissues and epiblast cells in vivo and in vitro. Here, we report for the first time the generation of Fgf5-P2A-Venus BAC transgenic (Tg) mice and show that the BAC Tg can recapitulate endogenous Fgf5 expression in epiblast and visceral endodermal cells of E6.5 and 7.5 embryos. We also show that Fgf5-P2A-Venus BAC Tg mEpiSCs in the undifferentiated state expressed abundant Venus, and upon reprogramming into naïve state, Venus was suppressed. Furthermore, while most Tg mEpiSCs expressed Venus abundantly, surprisingly the Tg mEpiSCs contained a minor subpopulation of Venus-negative cells that were capable of conversion to Venus-positive cells, indicating that even Fgf5 expression shows dynamic heterogeneity in mEpiSCs. Taken together, Fgf5-P2A-Venus BAC Tg mice and mEpiSCs generated in this study will be useful for developmental biology as well as stem cell biology research.
