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: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.
Project description:In mammals, retinal damage is followed by Müller glia cell activation and proliferation. While retinal gliosis persists in adult mammals after an insult or disease, some vertebrates, including zebrafish, have the capacity to regenerate. We believe we are the first group to show that gliosis is a fibrotic-like process in mammals’ eyes caused by differential activation of canonical and non-canonical TGFβ signaling pathways.
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.
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:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:Methoxyacetic acid (MAA) is the active metabolite of the widely used industrial chemical ethylene glycol monomethyl ether, an established testicular toxicant. MAA induces the degradation of testicular germ cells in association with changes in gene expression in both germ cells and Sertoli cells of the testis. This study investigates the impact of MAA on gene expression in testicular Leydig cells, which play a critical role in germ cell survival and male reproductive function. Cultured mouse TM3 Leydig cells were treated with MAA for 3, 8, and 24 h and global gene expression was monitored by microarray analysis. A total of 3,912 MAA-responsive genes were identified. Ingenuity Pathway analysis identified reproductive system disease, inflammatory disease and connective tissue disorder as the top biological functions affected by MAA. The MAA-responsive genes were classified into 1,366 early responders, 1,387 mid-responders, and 1,138 late responders, based on the time required for MAA to elicit a response. Analysis of enriched functional clusters for each subgroup identified 106 MAA early response genes involved in transcription regulation, including 32 genes associated with developmental processes and 60 DNA-binding proteins that responded to MAA rapidly but transiently, and which may contribute to the downstream effects of MAA seen for large numbers of mid and late response genes. Genes within the phosphatidylinositol/phospholipase C/calcium signaling pathway, whose activity is required for potentiation of nuclear receptor signaling by MAA, were also enriched in the set of early MAA response genes. These findings on the progressive changes in gene expression induced by MAA in Leydig cells may help elucidate the signaling pathways perturbed by this testicular toxicant and explain its mechanism of toxicity at the gene level.