Long-term effect on the transcriptome of a decrement in Norrin/Frizzled4/Lrp signaling in retinal endothelial cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE16703 | GEO | 2009/10/01
SECONDARY ACCESSION(S): PRJNA122803
REPOSITORIES: GEO
Similar Datasets
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.
2009-10-10 | E-GEOD-16703 | biostudies-arrayexpress
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
2009-10-10 | E-GEOD-16841 | biostudies-arrayexpress
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.
2009-10-01 | GSE16705 | GEO
Project description:Transcriptional profiles of the embryonic yolk sac from embryos with ectopic Norrin expression were compared to their wild type littermate controls. The goal is to identify the transcriptional response to Norrin-Frizzled 4 signaling during embryonic angiogenesis.
2009-10-01 | GSE16691 | GEO
Project description:Transcriptional profiles of the cerebellar endothelial cells from P16 Fz4-/- animals were compared to their wild type littermate controls. The goal is to characterize the long-term effect on the transcriptome of loss of Fz4 signaling in cerebellar endothelial cells. Endothelial cells were immuno-purification from P16 cerebellum using anti-PECAM antibody coated dynabeads. The cell immuno-purification procedure follows that described by Matsubara et al. (2000) and Su et al. (2003). 3 replicates of both wild type and Fz4-/- cerebellar endothelial cells were analyzed.
2009-10-01 | E-GEOD-16707 | biostudies-arrayexpress
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. Experiment Overall Design: To isolate the requisite retinal endothelial cell lines, we crossed Fz4-/- and control animals into the Immorto-mouse line in which a gamma interferon responsive H2Kb promoter directs the expression of a temperature sensitive SV40 large T-antigen (Jat et al., 1991). When cultured at 33°C in the presence of gamma interferon, this conditional oncogene system extends the proliferative capacity of a variety of cell types, which can be subsequently analyzed under phenotypically reverting conditions (37°C without gamma interferon). The cell immuno-purification procedure follows that described by Matsubara et al. (2000) and Su et al. (2003). The purified cells were then cultured and passaged at 33°C on dishes coated with 1% gelatin. For RNA preparation, cells were cultured at 37°C in the absence of interferon gamma. For the Matrigel experiments, endothelial cells previously starved for 24 hrs in DMEM with 1% FBS were trypsinized and plated on the Matrigel layer. The cells were incubated in DMEM with 1% FBS for 6 hours at 37°C before harvesting for RNA extraction. For the 293 cell co-culture experiments, endothelial cells were co-cultured with a human embryonic kidney (HEK) 293 cell line that stably expresses Norrin, or with control 293 cells.
2009-10-10 | E-GEOD-16705 | biostudies-arrayexpress
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
2014-06-05 | E-GEOD-56461 | biostudies-arrayexpress
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.
2014-06-05 | GSE56461 | GEO
Project description:The integrity of the blood–retina barrier (BRB) is crucial for phototransduction and vision, by tightly restricting transport of molecules between the blood and surrounding neuronal cells. Breakdown of the BRB leads to the development of retinal diseases. Here, we show that Netrin-1/Unc5b and Norrin/Lrp5 signaling establish a zonated endothelial cell gene expression program that controls BRB integrity. Using single-cell RNA sequencing (scRNA-seq) of postnatal BRB-competent mouse retina endothelial cells (ECs), we investigate >100 BRB genes encoding Wnt signaling components, tight junction proteins, and ion and nutrient transporters. We find that BRB gene expression is zonated across arteries, capillaries, and veins and regulated by opposing gradients of the Netrin-1 receptor Unc5b and Lrp5-β-catenin signaling between retinal arterioles and venules. Mice deficient for Ntn1 or Unc5b display more BRB leakage at the arterial end of the vasculature, while Lrp5 loss of function causes predominantly venular BRB leakage. ScRNA-seq of Ntn1 and Unc5b mutant ECs reveals down-regulated β-catenin signaling and BRB gene expression that is rescued by Ctnnb1 overactivation, along with BRB integrity. Mechanistically, we demonstrate that Netrin-1 and Norrin additively enhance β-catenin transcriptional activity and Lrp5 phosphorylation via the Discs large homologue 1 (Dlg1) scaffolding protein, and endothelial Lrp5-Unc5b function converges in protection of capillary BRB integrity. These findings explain how arteriovenous zonation is established and maintained in the BRB and reveal that BRB gene expression is regulated at the level of endothelial subtypes.
2024-12-03 | GSE282775 | GEO
Project description:Wnt signaling is initiated by Wnt ligand binding to the extracellular ligand binding domain, called the cysteine-rich domain (CRD), of a Frizzled (Fzd) receptor. Norrin, an atypical Fzd ligand, specifically interacts with Fzd4 to activate β-catenin-dependent canonical Wnt signaling. Much of the molecular basis that confers Norrin selectivity in binding to Fzd4 was revealed through the structural study of the Fzd4CRD-Norrin complex. However, how the ligand interaction, seemingly localized at the CRD, is transmitted across full-length Fzd4 to the cytoplasm remains largely unknown. Here, we show that a flexible linker domain, which connects the CRD to the transmembrane domain, plays an important role in Norrin signaling. The linker domain directly contributes to the high-affinity interaction between Fzd4 and Norrin as shown by ∼10-fold higher binding affinity of Fzd4CRD to Norrin in the presence of the linker. Swapping the Fzd4 linker with the Fzd5 linker resulted in the loss of Norrin signaling, suggesting the importance of the linker in ligand-specific cellular response. In addition, structural dynamics of Fzd4 associated with Norrin binding investigated by hydrogen/deuterium exchange MS revealed Norrin-induced conformational changes on the linker domain and the intracellular loop 3 (ICL3) region of Fzd4. Cell-based functional assays showed that linker deletion, L430A and L433A mutations at ICL3, and C-terminal tail truncation displayed reduced β-catenin-dependent signaling activity, indicating the functional significance of these sites. Together, our results provide functional and biochemical dissection of Fzd4 in Norrin signaling.
| S-EPMC6126767 | biostudies-literature