Project description:This SuperSeries is composed of the following subset Series:; GSE16691: Transcriptional regulation by Norrin-Frizzled4 signaling in the embryonic yolk sac; GSE16703: Long-term effect on the transcriptome of a decrement in Norrin/Frizzled4/Lrp signaling in retinal endothelial cells; GSE16705: Transcriptional response to Frizzled4 signaling in cultured retinal endothelial cells; GSE16707: Long-term effect on the transcriptome of loss of Frizzled4 signaling in cerebellar endothelial cells Experiment Overall Design: Refer to individual Series
Project description:To characterize the long-term effect on the transcriptome of a decrement in Norrin/Fz4/Lrp signaling, microarray hybridization was performed with RNA from acutely dissociated and anti-PECAM immunoaffinity-purified adult WT, Fz4-/-, Lrp5-/-, and Norrin- retinal vascular cells. Experiment Overall Design: Retinal endothelial cells from 5-10 adult mice were pooled for each sample, the cell immuno-purification procedure follows that described by Matsubara et al. (2000) and Su et al. (2003). 3 replicates of each mutant and 6 replicates of control wild type animals were analyzed.
Project description:To characterize the long-term effect on the transcriptome of a decrement in Norrin/Fz4/Lrp signaling, microarray hybridization was performed with RNA from acutely dissociated and anti-PECAM immunoaffinity-purified adult WT, Fz4-/-, Lrp5-/-, and Norrin- retinal vascular cells.
Project description:Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G-protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a co-activator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp co-receptor, and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. These experiments identify Gpr124 as a ligand-specific co-activator of canonical Wnt signaling. Total mRNA from HEK-293/STF cells was subjected to RNAseq
Project description:Using mice with targeted gene mutations, we identify (1) distinct roles for different canonical Wnt signaling components in central nervous system (CNS) vascular development and in the specification of the blood-brain and blood-retina barriers (BBB and BRB) and (2) differential sensitivities of the vasculature in various CNS regions to perturbations in canonical Wnt signaling components. We find nearly equivalent roles for Lrp5 and Lrp6 in brain vascular development and barrier maintenance but a dominant role for Lrp5 in the retinal vasculature, an especially high sensitivity of the BBB in the cerebellum and pons/interpeduncular nuclei to decrements in canonical Wnt signaling, and plasticity in the barrier properties of mature CNS vasculature. Brain and retinal vascular defects caused by loss of Norrin/Frizzled4 signaling can be fully rescued by stabilizing beta-catenin, and loss of beta-catenin’s transcriptional activation domain or expression of a dominant negative Tcf4 recapitulates the vascular development and barrier defects seen with loss of receptor, co-receptor, or ligand, indicating that Norrin/Frizzled4 signaling acts predominantly by beta-catenin-dependent transcriptional regulation. This work strongly supports a model in which identical or nearly identical canonical Wnt signaling mechanisms mediate neural tube and retinal vascularization and maintain the BBB and BRB. Total retina RNA from P10 WT, NdpKO, Ctnnb1flex3/+;Pdgfb-CreER, and NdpKO;Ctnnb1flex3/+;Pdgfb-CreER mice was subjected to RNAseq
Project description:Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G-protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a co-activator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp co-receptor, and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. These experiments identify Gpr124 as a ligand-specific co-activator of canonical Wnt signaling.
Project description:Transcriptional profiles of Fz4-/- retinal endothelial cells were compared to that of wild type endothelial cells under various culture conditions. The goal was to identify the transcriptional response to Frizzled 4 signaling in cultured retinal endothelial cells. To analyze the Norrin response of WT and Fz4-/- retinal endothelial cells in culture, we co-cultured these cells either with HEK293 cell line that stably expresses Norrin or with control 293 cells.