Development and Diversification of Retinal Amacrine Interneurons at Single Cell Resolution
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE12601 | GEO | 2009/06/01
SECONDARY ACCESSION(S): PRJNA112857
REPOSITORIES: GEO
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Project description: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.
2009-05-31 | E-GEOD-12601 | biostudies-arrayexpress
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.
2011-11-30 | E-GEOD-33088 | biostudies-arrayexpress
Project description:Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing (scRNA-seq) to profile >32,000 ACs from mice of both sexes, and applied computational methods to identify 63 AC types. We identified molecular markers for each type, and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types expressed markers for the canonical inhibitory neurotransmitters GABA or glycine, but several expressed neither or both. In addition, many expressed one or more neuropeptides, and two express glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological and morphological analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in mouse retina.
2020-06-22 | GSE149715 | GEO
Project description:The mammalian retina contains a complex mixture of different types of neurons. We find that the microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in the retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b, or the related miR-216a, can direct the formation of additional amacrine cells in the developing retina. In addition, we observe the loss of bipolar neurons in the retina after miR-216b expression. We identify the mRNA for the transcriptional regulator Foxn3 as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3 in the postnatal developing retina by RNAi also increases the formation of amacrine cells and reduces bipolar cell formation, while overexpression of Foxn3 inhibits amacrine cell formation prior to the expression of Ptf1a. Disruption of Foxn3 by CRISPR in embryonic retinal explants also reduces amacrine cell formation. Co-expression of Foxn3 partially reverses the effects of ectopic miR-216b on retinal cell type formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates expression of Foxn3 and other genes in amacrine cells.
2021-12-22 | GSE165827 | GEO
Project description:The mammalian retina contains a complex mixture of different types of neurons. We find that the microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in the retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b, or the related miR-216a, can direct the formation of additional amacrine cells in the developing retina. In addition, we observe the loss of bipolar neurons in the retina after miR-216b expression. We identify the mRNA for the transcriptional regulator Foxn3 as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3 in the postnatal developing retina by RNAi also increases the formation of amacrine cells and reduces bipolar cell formation, while overexpression of Foxn3 inhibits amacrine cell formation prior to the expression of Ptf1a. Disruption of Foxn3 by CRISPR in embryonic retinal explants also reduces amacrine cell formation. Co-expression of Foxn3 partially reverses the effects of ectopic miR-216b on retinal cell type formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates expression of Foxn3 and other genes in amacrine cells.
2021-12-22 | GSE165831 | GEO
Project description:The mammalian retina contains a complex mixture of different types of neurons. We find that the microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in the retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b, or the related miR-216a, can direct the formation of additional amacrine cells in the developing retina. In addition, we observe the loss of bipolar neurons in the retina after miR-216b expression. We identify the mRNA for the transcriptional regulator Foxn3 as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3 in the postnatal developing retina by RNAi also increases the formation of amacrine cells and reduces bipolar cell formation, while overexpression of Foxn3 inhibits amacrine cell formation prior to the expression of Ptf1a. Disruption of Foxn3 by CRISPR in embryonic retinal explants also reduces amacrine cell formation. Co-expression of Foxn3 partially reverses the effects of ectopic miR-216b on retinal cell type formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates expression of Foxn3 and other genes in amacrine cells.
2021-12-22 | GSE165830 | GEO
Project description:During development of the central nervous system (CNS), cycling uncommitted progenitor cells give rise to a variety of distinct neuronal and glial cell types. As these different cell types are born, they progress from newly specified cells to fully differentiated neurons and glia. In order to define the developmental processes of individual cell types, single cell expression profiling was carried out on developing ganglion and amacrine cells of the murine retina. Individual cells from multiple developmental stages were isolated and profiled on Affymetrix oligonucleotide arrays. These experiments have yielded an expanded view of the processes underway in developing retinal ganglion and amacrine cells, as well as several hundred new marker genes for these cell types. In addition, this study has allowed for the definition of some of the molecular heterogeneity both between developing ganglion and amacrine cells and among subclasses of each cell type. Keywords: Single retinal cell gene expression profiling across multiple mouse developmental stages
2007-12-31 | GSE9812 | GEO
Project description:During development of the central nervous system (CNS), cycling uncommitted progenitor cells give rise to a variety of distinct neuronal and glial cell types. As these different cell types are born, they progress from newly specified cells to fully differentiated neurons and glia. In order to define the developmental processes of individual cell types, single cell expression profiling was carried out on developing ganglion and amacrine cells of the murine retina. Individual cells from multiple developmental stages were isolated and profiled on Affymetrix oligonucleotide arrays. These experiments have yielded an expanded view of the processes underway in developing retinal ganglion and amacrine cells, as well as several hundred new marker genes for these cell types. In addition, this study has allowed for the definition of some of the molecular heterogeneity both between developing ganglion and amacrine cells and among subclasses of each cell type. 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. Cells were identified post hoc as either developing retinal ganglion cells, amacrine cells or rod photoreceptor cells.
2008-06-16 | E-GEOD-9812 | biostudies-arrayexpress
Project description:Visual processing depends on sensitive and balanced synaptic neurotransmission. Extracellular matrix proteins in the environment of cells are key modulators in synaptogenesis and synaptic plasticity. In the present study, we provide evidence that the combined loss of the four extracellular matrix components brevican, neurocan, tenascin-C and tenascin-R in quadruple knockout mice leads to severe retinal dysfunction and diminished visual motion processing in vivo. Remarkably, impaired visual motion processing was accompanied by a developmental loss of cholinergic direction-selective starburst amacrine cells. Additionally, we noted imbalance of inhibitory and excitatory synaptic signaling in the quadruple knockout retina. Collectively, the study offers novel insights into the functional importance of four key extracellular matrix proteins for retinal function, visual motion processing and synaptic signaling.
2023-12-18 | GSE230425 | GEO
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.
2011-11-30 | GSE33088 | GEO