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Long-term effect on the transcriptome of loss of Frizzled4 signaling in cerebellar endothelial cells

ABSTRACT: 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.

ORGANISM(S): Mus musculus

SUBMITTER: Xin Ye

PROVIDER: E-GEOD-16707 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.

Ye Xin X Wang Yanshu Y Cahill Hugh H Yu Minzhong M Badea Tudor C TC Smallwood Philip M PM Peachey Neal S NS Nathans Jeremy J

Cell 20091001 2

Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencin ...[more]

PMID: 19837032

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Transcription profiling of mouse decrement in Norrin/Frizzled4/Lrp signaling in retinal endothelial cells

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Transcription profiling of mouse Frizzled4 signaling in cultured retinal endothelial cells

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.

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