Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Development and Diversification of Retinal Amacrine Interneurons at Single Cell Resolution

ABSTRACT: The vertebrate retina uses diverse neuronal cell types arrayed into complex neural circuits to extract, process and relay information from the visual scene to the higher order processing centers of the brain. Amacrine cells, a diverse class of inhibitory interneurons, are thought to mediate the majority of the processing of the visual signal that occurs within the retina. Despite morphological characterization, the number of known molecular markers of amacrine cell types is still much smaller than the 26 morphological types that have been identified. Furthermore, it is not known how this diversity arises during development. Here, we have combined in vivo genetic labeling and single cell genome-wide expression profiling to: 1) Identify specific molecular types of amacrine cells; 2) Demonstrate the molecular diversity of the amacrine cell class. It is difficult to identify new markers of amacrine cells, due to the fact that they only comprise a small percentage of the total cells in the retina. Additionally, given that there are at least 26 distinct types of amacrine cells, population based approaches fail to achieve the precision necessary to discover markers of each type. To facilitate the identification of new markers for different amacrine cell classes and to more fully characterize the molecular signatures of these classes, we isolated single amacrine cells. To accomplish this goal, we introduced genetic reporters (pNdrg4::GFP or pSynapsin::GFP) into the developing retina (P0) by either in vivo or ex vivo electroporation. These reporters were observed to label morphologically distinct sets of amacrine cells at the different timepoints harvested in this study. Electroporated retinas were then dissociated at different time points and single retinal amacrine cells were isolated by virtue of their GFP expression and placed in tubes containing lysis buffer. Then, 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.

ORGANISM(S): Mus musculus

SUBMITTER: jeffrey trimarchi

PROVIDER: E-GEOD-12601 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Development and diversification of retinal amacrine interneurons at single cell resolution.

Cherry Timothy J TJ Trimarchi Jeffrey M JM Stadler Michael B MB Cepko Constance L CL

Proceedings of the National Academy of Sciences of the United States of America 20090526 23

The vertebrate retina uses diverse neuronal cell types arrayed into complex neural circuits to extract, process, and relay information from the visual scene to the higher order processing centers of the brain. Amacrine cells, a class of interneurons, are thought to mediate much of the processing of the visual signal that occurs within the retina. Although amacrine cells display extensive morphological diversity, the molecular nature of this diversity is largely unknown. Furthermore, it is not kn ...[more]

PMID: 19470466

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Project description:The development of complex tissues requires that mitotic progenitor cells integrate information from the environment. The highly varied outcomes of such integration processes undoubtedly depend at least in part upon variations among the gene expression programs of individual progenitor cells. To date, there has not been a comprehensive examination of these differences among progenitor cells of a particular tissue. Here, we used comprehensive gene expression profiling to define these differences among individual progenitor cells of the vertebrate retina. Retinal progenitor cells (RPCs) have been shown by lineage analysis to be multipotent throughout development and to produce distinct types of daughter cells in a temporal, conserved order. A total of 42 single RPCs were profiled on Affymetrix arrays. An extensive amount of heterogeneity in gene expression among RPCs, even among cells isolated from the same developmental time point, was observed. While many classes of genes displayed heterogeneity of gene expression, the expression of transcription factors constituted a significant amount of the observed heterogeneity. Additionally, the expression of cell cycle related transcripts showed differences among those associated with G2 and M, versus G1 and S phase, suggesting different levels of regulation for these genes. These data provide insights into the types of processes and genes that are fundamental to cell fate choices, proliferation decisions, and, for cells of the central nervous system, the underpinnings of the formation of complex circuitry. Experiment Overall Design: 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. A total of 42 retinal progenitors were identified post hoc and compared with ganglion, amacrine and photreceptor cells isolated from identical timepoints.

Project description:Brain circuits are assembled from a large variety of morphologically and functionally diverse cell types. It is not known how the intermingled cell types of individual brain regions differ in their expressed genomes. Here we describe an atlas of cell type transcriptomes of the adult retina. We found that each adult cell type expresses a specific set of genes, including a unique set of transcription factors, forming a “barcode” for cell identity. Cell type transcriptomes carry enough information to categorize cells into corresponding morphological classes and types. Surprisingly, several barcode genes are eye disease-associated genes that we demonstrate to be specifically expressed not only in photoreceptors but also in particular retinal circuit elements such as inhibitory neurons as well as in retinal microglia. Our data suggest that distinct cell types of individual brain regions are characterized by marked differences in their expressed genomes. To obtain insight into the developmental time course of adult cell type-specific gene expression, we followed gene expression in one amacrine cell group, Arc cells, for 20 postnatal days (P) starting at P1. Qualitative analysis of the expression time-course of cell type-specific genes revealed four patterns: increasing, decreasing or constant expression from P1 to P20, or a biphasic time-course (constant and increasing) with a switch at P9-P10. At this latter time point, bipolar cells begin to form synapses with amacrine and ganglion cells and the retina becomes light responsive. Pairwise correlations across the transcriptomes measured on each day exposed two major clusters, from P1 to P9 and from P10 to P20, which also suggested a transcriptional switch during the transition from P9 to P10. Thus, most adult cell type-specific genes are also expressed during development but at levels different to those in the adult. We performed gene expression analysis of one amacrine cell group, Arc, from P0 to P20 and for Starburst amacrine cells for the time point P3 and P18. All experiments were performed in biological triplicates. 1 retina was used for each time point and each sample of the biological triplicate.

Dataset Information

Development and Diversification of Retinal Amacrine Interneurons at Single Cell Resolution

Publications

Development and diversification of retinal amacrine interneurons at single cell resolution.

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