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Transcriptional profile changes in retinal pigment epithelial cell culture after treatment with an α7 nicotinic acetylcholine receptor agonist

ABSTRACT: Purpose: Eye drop application of PNU-282987, an α7 nAChR agonist, causes regeneration through development of Müller derived progeintor cells (MDPCs) that are generated as a result of signaling from activated retinal pigment epitheilal (RPE) cells . The goals of this study are to examine the transcrptional changes through RNA-seq after treatment with PNU-282987 in mammalian RPE cell culture that lead to the dedifferentation and generation of MDPCs in the adult mammalian retina. To validate the RNAseq findings, RT-qPCR was used. Finally, several genes that were identified were knocked out in RPE culture through a novel CRISPR/Cas12 system and the resulting activated supernatant was assayed for it's ability to cause neurogensis in the adult rodent retina. Methods: Retinal pigment epithelium (RPE-J) cells were treated with DMSO (vehicle control), nicotine (an α7 nAChR agonist that does not cause regeneration as a control), or PNU-282987, or MLA (an α7 nAChR antagonist as a control). The cells were treated for either 30 minutes, 1 hour, 3 hours, 8 hours, or 12 hours. RNA was extracted from RPE-J cells using Zymo Direct-zol RNA miniprep kit and mRNA proflies were generated by GeneWiz with the Illumina NextSeq 550 high-output platform. Results: Using an optimized data analysis workflow, GeneWiz mapped about 30 million sequence reads per sample to the rat genome (Rnor6.0) and identified over 15,000 transcripts. Deseq2 analysis was performed to determine log 2 fold changes in gene expression compared to DMSO control, nicotine control, and MLA control, and found over 450 significantly differentially expressed genes. Twelve genes were validated with qRT–PCR to compare trends and were found to be consistent. Eight genes identified through RNA-seq involved in inflammation response, receptor signaling, WNT signaling and early retinal development were knocked out using CRISPR/Cas12 in RPE-J culture. Intravitreal injections of KO lines supernatant after treatment with PNU-282987 was assayed for invovlment of these genes in the adult neuroregeneration response. Conclusions: PNU-282987 activation of RPE causes signficant transcript profile changes in RPE-J cell culture that leads to significant differentially expressed genes as seen in RNA-seq profiles and validated with qRT-PCR. Further, several genes identified in the RNA-seq were found to be necessary for the neurogenic response as seen with generation of KO lines of RPE. Our study is the first to show that activaiton of the α7 nAChR on RPE leads to genetic changes sufficient to induce adult mammalian neurogenesis in the rodent retina and that several novel genes are invovled in this pathway in mammals as compared to other vertebrate models of retinal regeneration.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE156758 | GEO | 2021/08/21

REPOSITORIES: GEO

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Project description:Purpose: Eye drop application of PNU-282987, an α7 nAChR agonist, causes regeneration through activation of Müller glia (MG) in the adult rodent retina. The goals of this study are to examine the transcrptional changes through RNA-seq after treatment with PNU-282987 in mammalian MG cell culture that result in dedifferentiated MG that have a retinal progentior cell(RPC)-like fate and to vallidate RNA-seq differentially expressed genes with qRT-PCR and immunocytochemistry. Methods: Retinal pigment epithelium (RPE-J) cells were treated with PNU-282987 for 24 hours and then washed extensivley. These treated RPE cells were co-cultured via a transwell system with MG (rMC-1) cell culture for 0, 12, 24 and 48 hours. RNA was extracted from MG using Zymo Direct-zol RNA miniprep kit and mRNA proflies were generated by GeneWiz with the Illumina NextSeq 550 high-output platform. Results: Using an optimized data analysis workflow, GeneWiz mapped about 30 million sequence reads per sample to the rat genome (Rnor6.0) and identified over 15,000 transcripts. Deseq2 analysis was performed to determine log 2 fold changes in gene expression compared to DMSO control and found 1050 significantly differentially expressed genes and 12 of these were validated with qRT–PCR to compare trends. Notably, the HB-EGF/Ascl1/Lin28 pathway was signficantly upregulated. ICC showed expression of RPC genes that were also seen in the RNA-seq profile. Conclusions: PNU-282987 activation of RPE causes signficant transcript profile changes in MG cell culture that leads to upregulation of the HB-EGF/Ascl1/Lin28 pathway that was seen with RNA-seq profiles and validated with qRT-PCR. Further, these gene expression changes lead to dedifferentation of MG to an RPC-like fate and expression of several retinal RPC markers were found in RNA-seq data set and was validated with immunocytochemistry. Our study is the first to show that activaiton of the α7 nAChR on RPE can lead to MG dedifferentiation and that the HB-EGF/Ascl1/Lin28 pathway is upregulated in these cells.

Project description:Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Keywords: Tissue interactions, retina, retinal pigment epithelium, blood-retinal barrier, tight junctions

Project description:Purpose: The purpose of this study was to develop a framework for analyzing RPE expression profiles from zebrafish eye mutants. Methods: The fish model we used was smarca4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4), a retinal dystrophic mutant that the retinal phenotype and expression profiles were previously characterized. Histological and Affymetrix GeneChip analyses were conducted to define the RPE defects and underlying differential expression respectively. Results: Histological analysis indicates that smarca4 RPE was formed but its differentiation was abnormal. In particular, ultra-structural analysis of smarca4 RPE by transmission electron microscopy showed a number of defects in melanogenesis, suggesting that the cytoskeletal dynamics was impaired. To compare the expression profile of normal wild-type (WT) and smarca4 RPE, their retinas and RPE-attached retinas were microdissected and the gene expression values of these tissues measured by Affymetrix GeneChip analysis. The RPE expression values were then estimated from these samples using an approach previously established by us. A factorial analysis was conducted using the expression values of RPE, retinal as well as the whole-embryo samples. Specific rules (contrasts) were built using the coefficients of the resulting fitted model to select for three groups of genes: 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina. The latter group consists of 39 genes that are highly related to cytoskeletal dynamics, melanogenesis, paracrine and intracellular signal transduction. Conclusions: Our analytical framework can potentially identify genes in zebrafish mutants that both retina and RPE are affected by the underlying mutation. The gene expression levels of three independent replicates of WT whole embryos (WA52), WT retinas (WR52), WT RPE-attached retinas (WRR52), smarca4/yng retinas (YR52), smarca4 RPE-attached retinas (YRR52) and smarca4 whole embryos (YA52) were measured by Affymetrix Zebrafish Whole Genome Arrays. All samples were collected at 52 hpf. The probe-level data of these sample groups were background adjusted, normalized and summarized by a robust multiarray average (RMA) algorithm. RPE expression values were estimated from the comparison between the retinal and RPE-attached retinal samples of the same genotype based on a method we previously developed (Leung et al., Invest Ophthalmol Vis Sci. 2007; 48:881). Then, a 3x2 factorial design was used to analyze the expression values of three kinds of tissue (T): whole embryo, retina and RPE in two kinds of mutation (M) background: WT and smarca4. Using the coefficients from the fitted models, contrasts were built to identify candidate genes that fulfilled specific criteria. The false discovery rate (FDR) q-value cutoff for a contrast was 0.001. Several contrasts were ultimately combined to allow for selection of genes that were regulated by Smarca4 in the retina and RPE. These include 1) Smarca4-regulated RPE genes, 2) Smarca4-regulated retinal genes, and 3) Smarca4-regulated RPE genes that are not differentially expressed in the retina.

Project description:Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes. Reprinted from <a href="https://www.ncbi.nlm.nih.gov/pubmed/?term=25446321">Whitmore, S.S., Wagner, A.H., DeLuca, A.P., Drack, A.V., Stone, E.M., Tucker, B.A., Zeng, S., Braun, T.A., Mullins, R.F., Scheetz, T.E., 2014. Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq. Experimental Eye Research</a>. Used under <a href="http://creativecommons.org/licenses/by-nc-sa/3.0/">Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported </a>license. Additional RNA sequencing was performed on temporal, macular, nasal, inferior, and superior retina from a fifth subject and is included in this dataset. See original publication for details.

Project description:Purpose: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier. Primary cultures of RPE can model the barrier, but are very sensitive to culture conditions. We examined how the neural retina regulates the RPE transcriptome in a culture model of embryonic development. Attention focused on the tight junctional genes essential for barrier function. Methods: Chick RPE from embryonic day 7 (E7) or embryonic day 14 (E14) was cultured on filters in a serum free medium. Media conditioned by organ culture of neural retinas was applied to the apical surface of the cultured RPE. Fetal bovine serum (2%) was included in some experiments. When the transepithelial electrical resistance (TER) reached a plateau, total RNA was isolated to probe the chick genome on Affymetrix microarrays. The expression tight junctional mRNAs was confirmed by real-time PCR and immunoblotting the protein. Results: The TER was slightly increased by serum, but increased 2-3X by retinal conditioned medium. Serum diminished the effect of retinal conditioned medium. In basal conditions, 86% of the transcriptome expressed during development in vivo was also expressed in culture. Approximately 5% of the transcriptome expressed in culture was absent in vivo. E14 retinal conditioned medium affected 15% of the transcriptome in E7 cultures (24% if serum was included), but only 1.9% in E14 cultures (12% with serum). Examination of 610 genes important for RPE function revealed that mRNAs for 17% were regulated by retinal conditioned medium alone in E7 cultures, compared to 6.2% for E14. For tight junctions, retinal conditioned medium had the most affect on members of the claudin family. These results were confirmed by quantitative real-time PCR. Besides regulating mRNA levels, immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization. Conclusions: Gene expression in primary cultures of embryonic RPE resembled the native tissue, but expression, and differentiation, is improved when elements of the normal extracellular environment are replicated in culture. For a small group of proteins, retinal secretions were required to maintain in vivo-like expression in culture. Albeit insufficient, retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE. Experiment Overall Design: RPE were isolated from chicken embryos on embryonic day 7 or 14 and cultured, as described <Rahner C, Fukuhara M, Peng S, Kojima S, Rizzolo LJ. The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci. 2004;117:3307-18>. E7 is when tight junctions of the RPE begin to form; E14 is when tight junctions achieve their mature morphological appearance in vivo. The cells were cultured in medium that contains or lacks E14 retinal conditioned medium. Because serum inhibits the effect of retinal conditioned medium, serum was added to some cultures, resulting in 4 culture conditions for each age. To isolate total RNA, the RNeasy Protect kit (Qiagen) was used according to the manufacturer's protocols. For each culture, 3 independent preparations were used for analysis on Affymetrix microarrays of the chicken genome (Santa Clara, CA). The quality of the total RNA was assessed by the Keck Center, Yale University using formamide gels and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA)

Project description:Purpose: The aim of this study was to give a comprehensive overview on spatial distribution of gene expression in the adult mouse retina and integrate this information into existing retinal gene expression databases. Methods: Total RNA was collected by laser capture microdissection from the ganglion cell layer, inner nuclear layer, photoreceptors and the retinal pigmented epithelium of adult mice and was analyzed by oligonucleotide microarrays. The results were validated by quantitative real time PCR and in situ hybridization. Results: The applied method resulted in good separation of cells of different retinal layers. The spatial distribution of gene expression was determined on a global scale in the retina and the RPE. Our results show good correlation with previously reported retinal gene expression and describe genes not yet characterized in the context of the retina. Conclusions: The complexity of the vertebrate retina makes it necessary to determine not only temporal but spatial distributions of gene expression .Our work expands the already significant but still incomplete knowledge of retinal gene expression and hopefully facilitates functional characterization of key factors of retinal development and maintenance. Laser capture microdissected regions of the adult mouse neuronal retina and retinal pigmented epithelium were subjected to microarray analysis. 5 conditions were investigated: ganglion cell layer (GCL, 3 biological replicates), innner nuclear layer (INL, 3 biological replicates), photoreceptor layer from Blk6 (PR-WT, 3 biological replicates) and NrlKO animals (PR-Nrl, 2 biological replicates) and retinal pigmented epithelium (RPE, 1 biological replicate). All samples are co-hybridized with a reference sample (retina). 2 or 3 technical replicates were used for each biological sample. technical replicate - labeled-extract: GC1-1, GC1-2, GC1-3 technical replicate - labeled-extract: GC2-1, GC2-2, GC2-3 technical replicate - labeled-extract: GCpool-1, GCpool-2, GCpool-3 technical replicate - labeled-extract: INL1-1, INL1-2, INL1-3 technical replicate - labeled-extract: INL2-1, INL2-2, INL2-3 technical replicate - labeled-extract: INLpool-1, INLpool-2, INLpool-3 technical replicate - labeled-extract: PR-Nrl1-1, PR-Nrl1-2 technical replicate - labeled-extract: PR-Nrl2-1, PR-Nrl2-2, PR-Nrl2-3 technical replicate - labeled-extract: PR-WT1-1, PR-WT1-2, PR-WT1-3 technical replicate - labeled-extract: PR-WT2-1, PR-WT2-2, PR-WT2-3 technical replicate - labeled-extract: PR-WTpool-1, PR-WTpool-2, PR-WTpool-3 technical replicate - labeled-extract: RPE-1, RPE-2, RPE-3

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Transcriptional profile changes in retinal pigment epithelial cell culture after treatment with an α7 nicotinic acetylcholine receptor agonist

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