Similar Datasets
Project description:Endothelial cells (ECs) display organ- and tissue-specific heterogeneity. In the eye, the retinal and choroidal vascular beds are distinct networks with different morphological and functional features with the former representing a tight barrier and the latter, a permeable fenestrated vasculature. Given that retinal health critically relies on the function of these vascular beds and that their dysfunction is implicated in a variety of retinal diseases, a molecular understanding of both physiological and pathophysiological characteristics of these distinct vasculatures is critical. Given their interspersed anatomic distribution among parenchymal cells, the study of EC gene expression, in vivo, has been hampered by the challenge of isolating pure populations of ocular ECs in sufficient quantities for large-scale transcriptomics. To address this challenge, we present a methodological and analytical workflow to facilitate inter-tissue comparisons of the in vivo EC translatome isolated from choroid, retina, and brain using the Cre-inducible NuTRAP flox construct and two widely-used endothelial cre mouse lines, constitutive Tie2-Cre and Tamoxifen-inducible Cdh5-CreERT2, which express EGFP and mCherry tags for cell-specific isolation of nuclei and ribosomes. Within each model, inter-tissue comparison of TRAP-RNAseq enrichment (TRAP translatome vs input transcriptome) showed tissue-specific gene enrichments with differential pathway representation. For each mouse model, inter-tissue comparison of the EC translatome (choroid vs brain, choroid vs retina, and brain vs retina) showed over 50% overlap of differentially expressed genes (DEGs) between the three paired comparisons, with differential pathway representation for each tissue. Pathway analysis of DEGs in the Cdh5-NT vs Tie2-NT comparison for retina, choroid, and brain predicted inhibition of processes related to myeloid cell function and activation, consistent with more specific targeting of ECs in the Cdh5-NT than in the Tie2-NT model. While TRAP enriches for EC transcripts in both models, it also captures myeloid transcripts in the Tie2-NT model. We confirmed these observations with a cell sorting approach. We suggest experimental/analytical considerations should be taken when selecting cre-lines to target ECs.
2024-12-31 | GSE270200 | GEO
Project description:Endothelial cells (ECs) display organ- and tissue-specific heterogeneity. In the eye, the retinal and choroidal vascular beds are distinct networks with different morphological and functional features with the former representing a tight barrier and the latter, a permeable fenestrated vasculature. Given that retinal health critically relies on the function of these vascular beds and that their dysfunction is implicated in a variety of retinal diseases, a molecular understanding of both physiological and pathophysiological characteristics of these distinct vasculatures is critical. Given their interspersed anatomic distribution among parenchymal cells, the study of EC gene expression, in vivo, has been hampered by the challenge of isolating pure populations of ocular ECs in sufficient quantities for large-scale transcriptomics. To address this challenge, we present a methodological and analytical workflow to facilitate inter-tissue comparisons of the in vivo EC translatome isolated from choroid, retina, and brain using the Cre-inducible NuTRAP flox construct and two widely-used endothelial cre mouse lines, constitutive Tie2-Cre and Tamoxifen-inducible Cdh5-CreERT2, which express EGFP and mCherry tags for cell-specific isolation of nuclei and ribosomes. Within each model, inter-tissue comparison of TRAP-RNAseq enrichment (TRAP translatome vs input transcriptome) showed tissue-specific gene enrichments with differential pathway representation. For each mouse model, inter-tissue comparison of the EC translatome (choroid vs brain, choroid vs retina, and brain vs retina) showed over 50% overlap of differentially expressed genes (DEGs) between the three paired comparisons, with differential pathway representation for each tissue. Pathway analysis of DEGs in the Cdh5-NT vs Tie2-NT comparison for retina, choroid, and brain predicted inhibition of processes related to myeloid cell function and activation, consistent with more specific targeting of ECs in the Cdh5-NT than in the Tie2-NT model. While TRAP enriches for EC transcripts in both models, it also captures myeloid transcripts in the Tie2-NT model. We confirmed these observations with a cell sorting approach. We suggest experimental/analytical considerations should be taken when selecting cre-lines to target ECs.
2024-12-31 | GSE270197 | GEO
Project description:Endothelial cells (ECs) display organ- and tissue-specific heterogeneity. In the eye, the retinal and choroidal vascular beds are distinct networks with different morphological and functional features with the former representing a tight barrier and the latter, a permeable fenestrated vasculature. Given that retinal health critically relies on the function of these vascular beds and that their dysfunction is implicated in a variety of retinal diseases, a molecular understanding of both physiological and pathophysiological characteristics of these distinct vasculatures is critical. Given their interspersed anatomic distribution among parenchymal cells, the study of EC gene expression, in vivo, has been hampered by the challenge of isolating pure populations of ocular ECs in sufficient quantities for large-scale transcriptomics. To address this challenge, we present a methodological and analytical workflow to facilitate inter-tissue comparisons of the in vivo EC translatome isolated from choroid, retina, and brain using the Cre-inducible NuTRAP flox construct and two widely-used endothelial cre mouse lines, constitutive Tie2-Cre and Tamoxifen-inducible Cdh5-CreERT2, which express EGFP and mCherry tags for cell-specific isolation of nuclei and ribosomes. Within each model, inter-tissue comparison of TRAP-RNAseq enrichment (TRAP translatome vs input transcriptome) showed tissue-specific gene enrichments with differential pathway representation. For each mouse model, inter-tissue comparison of the EC translatome (choroid vs brain, choroid vs retina, and brain vs retina) showed over 50% overlap of differentially expressed genes (DEGs) between the three paired comparisons, with differential pathway representation for each tissue. Pathway analysis of DEGs in the Cdh5-NT vs Tie2-NT comparison for retina, choroid, and brain predicted inhibition of processes related to myeloid cell function and activation, consistent with more specific targeting of ECs in the Cdh5-NT than in the Tie2-NT model. While TRAP enriches for EC transcripts in both models, it also captures myeloid transcripts in the Tie2-NT model. We confirmed these observations with a cell sorting approach. We suggest experimental/analytical considerations should be taken when selecting cre-lines to target ECs.
2024-12-31 | GSE270198 | GEO
Project description:Comparative Analysis of In vivo Endothelial Cell Translatomes Across Central Nervous System Vascular Beds
| PRJNA1125395 | ENA
Project description:Comparative Analysis of In vivo Endothelial Cell Translatomes Across Central Nervous System Vascular Beds (Cdh5)
| PRJNA1125403 | ENA
Project description:Comparative Analysis of In vivo Endothelial Cell Translatomes Across Central Nervous System Vascular Beds (Tie2)
| PRJNA1125400 | ENA
Project description:Comparative Analysis of In vivo Endothelial Cell Translatomes Across Central Nervous System Vascular Beds (EC sorted)
| PRJNA1125399 | ENA
Project description:Summary Cell-specific RNA sequencing has revolutionized the study of cell biology. Here, we present a protocol to assess cell-specific translatomes of genetically targeted cell types. We focus on astrocytes and describe RNA purification using RiboTag tools. Unlike single-cell RNA sequencing, this approach allows high sequencing depth to detect low expression genes, and the exploration of RNAs translated in subcellular compartments. Furthermore, it avoids underestimation of transcripts from cells susceptible to cell isolation procedures. The protocol can be applied to a variety of cell types. For complete details on the use and execution of this protocol, please refer to Chai et al. (2017), Díaz-Castro et al. (2021), Díaz-Castro et al. (2019), Srinivasan et al. (2016), and Yu et al. (2018). Graphical abstract Highlights • A protocol for investigating cell-specific translatomes in mouse brain cells• Purification of RNA from genetically targeted cells using RiboTag tools• Recommended antibodies and oligonucleotides to assess brain-cell specificity• Sample quality assessment tips for generating the most reliable data Cell-specific RNA sequencing has revolutionized the study of cell biology. Here, we present a protocol to assess cell-specific translatomes of genetically targeted cell types. We focus on astrocytes and describe RNA purification using RiboTag tools. Unlike single-cell RNA sequencing, this approach allows high sequencing depth to detect low expression genes, and the exploration of RNAs translated in subcellular compartments. Furthermore, it avoids underestimation of transcripts from cells susceptible to cell isolation procedures. The protocol can be applied to a variety of cell types.
| S-EPMC9127423 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2019-10-10 | GSE138630 | GEO
Project description:Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.
| S-EPMC6876253 | biostudies-literature