Analysis of gene expression in wildtype and Notch1 mutant retinal cells by single cell profiling
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE35682 | GEO | 2013/09/06
SECONDARY ACCESSION(S): PRJNA152407
REPOSITORIES: GEO
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Project description:Loss of Notch1 in retinal progenitor cells (RPCs) during postnatal retinal development results in the overproduction of rod photoreceptors at the expense of interneurons and glia. To examine the molecular underpinnings of this observation, microarray analysis of singla retinal cells from wildtype (WT) or Notch1 conditional knockout (N1-CKO) retinas was performed. The majority of N1-CKO cells lost expression of known Notch target genes. These cells also had low levels of RPC and cell cycle genes, and robustly upregulated rod precursor genes. In addition, single WT cells, in which cell cycle marker genes were downregulated, expressed markers of both rod photoreceptors and interneurons. These results demonstrate that individual, newly postmitotic retinal cells can begin to differentiate into more than one cell type, and that this transitional state may be dependent on Notch1 signaling. We used microarrays to examine gene expression changes that occur in single cells after the removal of Notch1 during retinal development. Retinas of Notch1fl/fl P0 pups were electroporated in vivo with plamids encoding Cre driven by a broadly active promoter, CAG, along with a Cre-responsive GFP reporter, also driven by CAG promoter (CALNL-GFP). For controls, the retinas of sibling Notch1fl/fl pups were electroporated with CAG:GFP. The animals were sacrificed at P3 to allow time for Notch1 to be genetically removed and downstream gene expression changes to occur. Retinas electroporated with either CAG;Cre and CALNL-GFP or CAG:GFP alone were dissected and dissociated to individual cells, which were harvested under a dissecting microscope on the basis of their GFP signal.
2013-09-06 | E-GEOD-35682 | biostudies-arrayexpress
Project description:Previous lineage analyses have shown that retinal progenitor cells (RPCs) are multipotent throughout development, and expression profiling studies have shown a great deal of molecular heterogeneity among RPCs. To determine if the molecular heterogeneity predicts that an RPC will produce particular types of progeny, clonal lineage analysis was used to investigate the progeny of a subset of RPCs, those that express the basic helix-loop-helix (bHLH) transcription factor, Olig2. In contrast to the large and complex set of clones generated by viral marking of random embryonic RPCs, the embryonic Olig2+ RPCs underwent terminal divisions, producing small clones with primarily two of the five cell types being made by the pool of RPCs at that time. The embryonically produced cell types made by Olig2+ RPCs were cone photoreceptors and horizontal cell (HC) interneurons. Moreover, the embryonic Olig2+ RPC did not make the later Olig2+ RPC. The later, postnatal Olig2+ RPCs also made terminal divisions, which were biased towards production of rod photoreceptors and amacrine cell (AC) interneurons. These data indicate that the multipotent progenitor pool is made up of distinctive types of RPCs, which have biases towards producing subsets of retinal neurons in a terminal division, with the types of neurons produced varying over time. This strategy is similar to that of the developing Drosophila melanogaster ventral nerve cord, with the Olig2+ cells behaving as ganglion mother cells. Single retinal cells were isolated in tubes containing lysis buffer, their mRNAs were reverse transcribed, and the resulting cDNAs were PCR amplified for 35 cycles. Labeled cDNA samples were hybridized to Affymetrix 430 2.0 microarrays and the data was normalized using MAS5.0 software. These cells were examined for the expression of Olig2 or other bHLH factors.
2012-04-12 | E-GEOD-37207 | biostudies-arrayexpress
Project description:Previous lineage analyses have shown that retinal progenitor cells (RPCs) are multipotent throughout development, and expression profiling studies have shown a great deal of molecular heterogeneity among RPCs. To determine if the molecular heterogeneity predicts that an RPC will produce particular types of progeny, clonal lineage analysis was used to investigate the progeny of a subset of RPCs, those that express the basic helix-loop-helix (bHLH) transcription factor, Olig2. In contrast to the large and complex set of clones generated by viral marking of random embryonic RPCs, the embryonic Olig2+ RPCs underwent terminal divisions, producing small clones with primarily two of the five cell types being made by the pool of RPCs at that time. The embryonically produced cell types made by Olig2+ RPCs were cone photoreceptors and horizontal cell (HC) interneurons. Moreover, the embryonic Olig2+ RPC did not make the later Olig2+ RPC. The later, postnatal Olig2+ RPCs also made terminal divisions, which were biased towards production of rod photoreceptors and amacrine cell (AC) interneurons. These data indicate that the multipotent progenitor pool is made up of distinctive types of RPCs, which have biases towards producing subsets of retinal neurons in a terminal division, with the types of neurons produced varying over time. This strategy is similar to that of the developing Drosophila melanogaster ventral nerve cord, with the Olig2+ cells behaving as ganglion mother cells.
2012-04-13 | GSE37207 | GEO
Project description:We report RNAseq analysis of the transcriptome of retinas from mature rod-specific Dicer1 cKO mice and control littermates lacking Cre expression in order to better understand changes in gene regulation that could lead to retinal degeneration in cKO mice. Examine retinal transcriptome of 3 biological replicates for each genotype from 4-week-old animals with tissue collected between 8:00 - 10:00AM
2014-08-14 | E-GEOD-55377 | biostudies-arrayexpress
Project description:We report RNAseq analysis of the transcriptome of retinas from mature rod-specific Dicer1 cKO mice and control littermates lacking Cre expression in order to better understand changes in gene regulation that could lead to retinal degeneration in cKO mice. Examine retinal transcriptome of 3 biological replicates for each genotype from 4-week-old animals with tissue collected between 8:00 - 10:00AM
2014-08-14 | E-GEOD-55376 | biostudies-arrayexpress
Project description:To comprehensively profile cell types in the human retina, we performed single cell RNA-sequencing on 20,009 cells obtained post-mortem from three donors and compiled a reference transcriptome atlas. Using unsupervised clustering analysis, we identified 18 transcriptionally distinct clusters representing all known retinal cells: rod photoreceptors, cone photoreceptors, Müller glia cells, bipolar cells, amacrine cells, retinal ganglion cells, horizontal cells, retinal astrocytes and microglia.
2019-08-21 | E-MTAB-7316 | biostudies-arrayexpress
Project description:To better understand the mechanistic basis of aging and its relationship with retinal degeneration, we examined gene expression changes in aging rod photoreceptors. Rod photoreceptor cell death is a feature of normal retinal aging and is accelerated in many retinal degenerative diseases, including AMD, the leading cause of untreatable adult blindness in the United States and other western countries. To our knowledge, the examination of age-related gene expression changes in a specific neuronal cell-type is novel, and it has allowed us to identify significant age-related changes with better resolution than is possible with whole retina samples. We used flow cytometry and a transgenic mouse with GFP-tagged rod photoreceptors to purify this specific cell population, and gene expression changes were evaluated at three time points using microarrays and quantitative RT-PCR. Our results suggest that aging is progressive, beginning even in young adult mice. Although rod photoreceptors are highly specialized neurons, our analyses revealed changes in consensus pathways of aging, including oxidative phosphorylation and stress responses affecting transcription and inflammation. In addition, we identified stress response processes that may be especially relevant for the aging retina and retinal diseases, such as angiogenesis and nuclear receptor signaling pathways that affect retinoid and lipid metabolism. We used flow cytometry and a transgenic mouse with GFP-tagged rod photoreceptors to purify this specific cell population, and gene expression changes were evaluated at three time points using microarrays and quantitative RT-PCR.
2010-06-14 | E-GEOD-22317 | biostudies-arrayexpress
Project description:Mutation of rod photoreceptor-enriched transcription factors is a major cause of inherited blindness. We identified the orphan nuclear hormone receptor ERRβ as selectively expressed in rod photoreceptors. Overexpression of ERRβ induces expression of rod-specific genes in retinas of both wildtype and in Nrl-/- mice, which lack rod photoreceptors. Mutation of ERRβ results in dysfunction and degeneration of rods, while inverse agonists of ERRβ trigger rapid rod degeneration, which is rescued by constitutively active mutants of ERRβ. ERRβ coordinates expression of multiple genes that are rate-limiting regulators of ATP generation and consumption in photoreceptors. Furthermore, enhancing ERRβ activity rescues photoreceptor defects that result from loss of the photoreceptor-specific transcription factor Crx. Our findings demonstrate that ERRβ is a critical regulator of rod photoreceptor function and survival, and suggest that ERRβ agonists may be useful in the treatment of certain retinal dystrophies. Affymetrix MOE430 microarrays were used to analyze the expression patterns of P21 mouse retinal tissues. The results were compared across the variable of Genotype, specifically ERRβ knockout versus wildtype.
2010-07-30 | E-GEOD-21944 | biostudies-arrayexpress
Project description:Photoreceptor disorders are collectively known as retinal degeneration (RD), and include retinitis pigmentosa (RP), cone-rod dystrophy and age related macular degeneration (AMD). These disorders are largely genetic in origin; individual mutations in any one of >200 genes cause RD, making mutation specific therapies prohibitively expensive. A better treatment plan, particularly for late stage disease, may involve stem cell transplants into the photoreceptor or ganglion cell layers of the retina. Stem cells from young mouse retinas can be transplanted, and can form photoreceptors in adult retinas. These cells can be grown in tissue culture, but can no longer form photoreceptors. We have used microarrays to investigate differences in gene expression between cultured retinal progenitor cells (RPCs) that have lost photoreceptor potential, postnatal day 1 (pn1) retinas and the postnatal day 5 (pn5) retinas that contain transplantable photoreceptors. We have also compared FACS sorted Rho-eGFP expressing rod photoreceptors from pn5 retinas with Rho-eGFP negative cells from the same retinas. We have identified over 300 genes upregulated in rod photoreceptor development in multiple comparisons, 37 of which have been previously identified as causative of retinal disease when mutated. It is anticipated that this research should bring us closer to growing photoreceptors in culture and therefore better treatments for RD. This dataset is also a resource for those seeking to identify novel retinopathy genes in RD patients. We extracted whole retinas from postnatal day 1 (Pn1) and postnatal day 5 (Pn5) mice, and compared them with cultured RPCs derived from pn5 retinas, using Affymetrix mouse 430A_2 arrays. We also extracted cells from Rho-eGFP Pn5 retinas and FACS sorted them. GFP+ve cells represent immature rod photoreceptors, as they express the Rho-eGFP fusion protein, which is only expressed in rods. GFP-ve cells represent all other retinal neurons. These samples were amplified and compared using Affymetrix mouse 430A_2arrays, by Source Biosciences GMBH, Berlin, Germany. Results from immature rods were then compared with those from other retinal neurons, while results from whole Pn5 retinas were compared with Pn1 retinas (which don't yet express rod specific genes), and RPCs, which are glial precursors. RPCs were also compared with Pn1 retinas. Genes which showed changed expression profiles in at least 3/4 of comparisons were prioritised for further investigation.
2015-01-20 | E-GEOD-59201 | biostudies-arrayexpress
Project description:Generation of three-dimensional (3D) organoids with optic cup like structures from pluripotent stem cells has created opportunities for investigating mammalian retinal development in vitro. However, retinal organoids in culture do not completely reflect the developmental state and in vivo architecture of the rod-dominant mouse retina. To assess rod photoreceptor differentiation in retinal organoids, we took advantage of Nrl-GFP mice that show rod-specific expression of GFP directed by the promoter of leucine zipper transcription factor NRL. Using embryonic and induced pluripotent stem cells (ESCs and iPSCs, respectively) derived from the Nrl-GFP mouse, we were successful in establishing long-term retinal organoid cultures (up to day 35) using modified culture conditions (called High Efficiency Hypoxia Induced Generation of Photoreceptors in Retinal Organoids, or HIPRO). We demonstrate efficient differentiation of pluripotent stem cells to retinal structures, with over 70% of embryoid bodies forming optic vesicles at Day (D)7, >50% producing optic cups by D10, and a majority of these surviving until at least D35. The HIPRO organoids include distinct inner retina neurons in somewhat stratified architecture and mature Müller glia spanning the entire retina. Almost 70% of the cells in retinal organoids are rod photoreceptors that exhibit elongated cilia. Transcriptome profiles of GFP+ rod photoreceptors, purified from organoids at Day 25-35, demonstrate a high correlation to gene profiles of purified rods from mouse retina at postnatal day (P) 2 to 6, indicating their early state of differentiation. Our 3D retinal organoids thus closely mimic in vivo retinogenesis and provide an efficient in vitro model to investigate photoreceptor development and modeling disease pathology.
2016-09-09 | GSE86790 | GEO