scholarly journals Molecular identification of sixty-three amacrine cell types completes a mouse retinal cell atlas

2020 ◽  
Author(s):  
Wenjun Yan ◽  
Mallory A. Laboulaye ◽  
Nicholas M. Tran ◽  
Irene E. Whitney ◽  
Inbal Benhar ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
High Throughput ◽  
Amacrine Cells ◽  
Cell Types ◽  
Retinal Cell ◽  
Mouse Retina ◽  
Single Cell Rna Sequencing ◽  
Neuronal Types ◽  
The Brain

ABSTRACTAmacrine 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 mouse retina, 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 express markers for the canonical inhibitory neurotransmitters GABA or glycine, but several express neither or both. In addition, many express 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.SIGNIFICANCE STATEMENTThe mouse retina is a leading model for analyzing the development, structure, function and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing (scRNA-seq) to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for use of multiple neurotransmitters and neuropeptides and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains 130 neuronal types, and is therefore comparable in complexity to other regions of the brain.

scholarly journals Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals putative regulatory toggles operating in native and iPS-derived retina

2020 ◽  
Author(s):  
Anouk Georges ◽  
Haruko Takeda ◽  
Arnaud Lavergne ◽  
Michiko Mandai ◽  
Fanny Lepiemme ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Retinal Development ◽  
Amacrine Cells ◽  
Regulatory Elements ◽  
Precursor Cells ◽  
Retinal Cell ◽  
Rna Seq ◽  
Combined Analysis ◽  
Temporal Sampling

AbstractBackgroundIt has recently become possible to recapitulate retinal development from induced pluripotent stem cells, opening new investigative and therapeutic opportunities. Single cell RNA sequencing allows comparison of transcriptome unfolding during in vivo and in vitro development at single cell resolution, which can be integrated with information about accessible regulatory elements identified by ATAC-Seq.ResultsWe report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from native and iPSC-derived murine retina at four matched developmental stages spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, and RNA velocity to show that iPSC-derived 3D retinal aggregates broadly recapitulate the native developmental trajectories with evidence supporting re-specification from amacrine cells to horizontal and photoreceptor precursor cells, as well as a direct differentiation of Tbr1+ retinal ganglion cells from neuro-epithelium cells. We show relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in iPSC-derived retina. We generate bulk ATAC-Seq data for native and iPSC-derived murine retina identifying ∼125,000 peaks. We combine single-cell RNA-Seq with ATAC-Seq information and obtain evidence that approximately halve the transcription factors that are dynamically regulated during retinal development may act as repressors rather than activators. We propose that sets of activators and repressors with cell-type specific expression control “regulatory toggles” that lock cells in distinct transcriptome states underlying differentiation, with subtle but noteworthy differences between native and iPSC-derived retina.ConclusionsCombined analysis of single-cell RNA-Seq and ATAC-Seq information has refined the comparison of native and iPS-derived retinal development.

scholarly journals Targeted single-cell RNA sequencing of transcription factors enhances the identification of cell types and trajectories

2021 ◽  
Author(s):  
Alexandra Pokhilko ◽  
Adam E. Handel ◽  
Fabiola Curion ◽  
Viola Volpato ◽  
Emma S. Whiteley ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Single Cell Rna Sequencing

scholarly journals Single-Cell Regulatory Network Inference and Clustering Identifies Cell-Type Specific Expression Pattern of Transcription Factors in Mouse Sciatic Nerve

2021 ◽  
Vol 15 ◽  
Author(s):  
Mingchao Li ◽  
Qing Min ◽  
Matthew C. Banton ◽  
Xinpeng Dun
Keyword(s):  
Transcription Factors ◽  
Sciatic Nerve ◽  
Single Cell ◽  
Cell Types ◽  
Cell Type ◽  
Specific Expression ◽  
Single Cell Rna Sequencing ◽  
Cell Type Specific Expression ◽  
Cell Type Specific ◽  
Different Cell Types

Advances in single-cell RNA sequencing technologies and bioinformatics methods allow for both the identification of cell types in a complex tissue and the large-scale gene expression profiling of various cell types in a mixture. In this report, we analyzed a single-cell RNA sequencing (scRNA-seq) dataset for the intact adult mouse sciatic nerve and examined cell-type specific transcription factor expression and activity during peripheral nerve homeostasis. In total, we identified 238 transcription factors expressed in nine different cell types of intact mouse sciatic nerve. Vascular smooth muscle cells have the lowest number of transcription factors expressed with 17 transcription factors identified. Myelinating Schwann cells (mSCs) have the highest number of transcription factors expressed, with 61 transcription factors identified. We created a cell-type specific expression map for the identified 238 transcription factors. Our results not only provide valuable information about the expression pattern of transcription factors in different cell types of adult peripheral nerves but also facilitate future studies to understand the function of key transcription factors in the peripheral nerve homeostasis and disease.

scholarly journals High-throughput single-cell transcriptome profiling of plant cell types

2018 ◽  
Author(s):  
Christine N. Shulse ◽  
Benjamin J. Cole ◽  
Gina M. Turco ◽  
Yiwen Zhu ◽  
Siobhan M. Brady ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Transcriptome Profiling ◽  
Cell Types ◽  
Marker Genes ◽  
Single Cell Rna Sequencing ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome ◽  
Heterogeneous Tissues ◽  
Genomic Basis

AbstractSingle-cell transcriptome analysis of heterogeneous tissues can provide high-resolution windows into the genomic basis and spatiotemporal dynamics of developmental processes. Here we demonstrate the feasibility of high-throughput single-cell RNA sequencing of plant tissue using the Drop-seq approach. Profiling of >4,000 individual cells from the Arabidopsis root provides transcriptomes and marker genes for a diversity of cell types and illuminates the gene expression changes that occur across endodermis development.

scholarly journals Multiscale Co-Expression in the Brain

2020 ◽  
Author(s):  
Benjamin D. Harris ◽  
Megan Crow ◽  
Stephan Fischer ◽  
Jesse Gillis
Keyword(s):  
Motor Cortex ◽  
Single Cell ◽  
Rna Sequencing ◽  
Primary Motor Cortex ◽  
Cell Types ◽  
Cell Type ◽  
Single Cell Rna Sequencing ◽  
Cell Type Specific ◽  
The Brain ◽  
Regulatory Landscape

ABSTRACTSingle-cell RNA-sequencing (scRNAseq) data can reveal co-regulatory relationships between genes that may be hidden in bulk RNAseq due to cell type confounding. Using the primary motor cortex data from the Brain Initiative Cell Census Network (BICCN), we study cell type specific co-expression across 500,000 cells. Surprisingly, we find that the same gene-gene relationships that differentiate cell types are evident at finer and broader scales, suggesting a consistent multiscale regulatory landscape.

scholarly journals Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and cis-regulatory modules in the retina

Development ◽  
2020 ◽  
Vol 147 (14) ◽  
pp. dev187922 ◽  
Author(s):  
Candace S. Y. Chan ◽  
Nicolas Lonfat ◽  
Rong Zhao ◽  
Alexander E. Davis ◽  
Liang Li ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Cell Types ◽  
Retinal Cell ◽  
Mouse Retina ◽  
Cell Type ◽  
Specific Expression ◽  
Regulatory Modules ◽  
A Cell

ABSTRACTTranscription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the mouse retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs, respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells use a cohort of TFs with different expression patterns and multiple CRMs with different chromatin configurations to regulate the expression of Otx2 precisely.

scholarly journals Generation of human neural retina transcriptome atlas by single cell RNA sequencing

2018 ◽  
Author(s):  
Samuel W. Lukowski ◽  
Camden Y. Lo ◽  
Alexei Sharov ◽  
Quan H. Nguyen ◽  
Lyujie Fang ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Amacrine Cells ◽  
Cell Types ◽  
Muller Glia ◽  
Müller Glia ◽  
Cone Photoreceptors ◽  
Rod Photoreceptors ◽  
Retinal Cells ◽  
Single Cell Rna Sequencing

SummaryThe retina is a highly specialized neural tissue that senses light and initiates image processing. Although the functional organisation of specific cells within the retina has been well-studied, the molecular profile of many cell types remains unclear in humans. 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. Notably, our data captured molecular profiles for healthy and early degenerating rod photoreceptors, and revealed a novel role of MALAT1 in putative rod degeneration. We also demonstrated the use of this retina transcriptome atlas to benchmark pluripotent stem cell-derived cone photoreceptors and an adult Müller glia cell line. This work provides an important reference with unprecedented insights into the transcriptional landscape of human retinal cells, which is fundamental to our understanding of retinal biology and disease.

scholarly journals Comprehensive analysis of a mouse model of spontaneous uveoretinitis using single-cell RNA sequencing

2019 ◽  
Vol 116 (52) ◽  
pp. 26734-26744 ◽  
Author(s):  
Jacob S. Heng ◽  
Sean F. Hackett ◽  
Genevieve L. Stein-O’Brien ◽  
Briana L. Winer ◽  
John Williams ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Rna Sequencing ◽  
Expression Patterns ◽  
Cell Types ◽  
Retinal Cell ◽  
Central Tolerance ◽  
Single Cell Rna Sequencing ◽  
Unbiased Manner ◽  
Lymphoid Structures

Autoimmune uveoretinitis is a significant cause of visual loss, and mouse models offer unique opportunities to study its disease mechanisms.Aire−/−mice fail to express self-antigens in the thymus, exhibit reduced central tolerance, and develop a spontaneous, chronic, and progressive uveoretinitis. Using single-cell RNA sequencing (scRNA-seq), we characterized wild-type andAire−/−retinas to define, in a comprehensive and unbiased manner, the cell populations and gene expression patterns associated with disease. Based on scRNA-seq, immunostaining, and in situ hybridization, we infer that 1) the dominant effector response inAire−/−retinas is Th1-driven, 2) a subset of monocytes convert to either a macrophage/microglia state or a dendritic cell state, 3) the development of tertiary lymphoid structures constitutes part of theAire−/−retinal phenotype, 4) all major resident retinal cell types respond to interferon gamma (IFNG) by changing their patterns of gene expression, and 5) Muller glia up-regulate specific genes in response to IFN gamma and may act as antigen-presenting cells.

scholarly journals Single-cell molecular and cellular architecture of the mouse neurohypophysis

2019 ◽  
Author(s):  
Qiyu Chen ◽  
Dena Leshkowitz ◽  
Janna Blechman ◽  
Gil Levkowitz
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Adult Male ◽  
Spatial Organization ◽  
Cell Types ◽  
Intermediate Lobe ◽  
Posterior Lobe ◽  
Single Cell Rna Sequencing ◽  
The Brain

AbstractThe neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin and arginine-vasopressin from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single cell RNA sequencing, we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell-types serving as a valuable resource for further functional research.Significance StatementThe neurohypophysis (NH) is a major neuroendocrine interface, which allows the brain to regulate the function of peripheral organs in response to specific physiological demands. Despite its importance, a comprehensive molecular description of cell identities in the NH is still lacking. Utilizing single cell RNA sequencing technology, we identified the transcriptomes of five major neurohypophyseal cell types in the adult male mice and mapped the spatial distribution of selected cell types in situ. We revealed an unexpected cellular heterogeneity of the neurohypophysis and provide novel molecular markers for neurohypophyseal cell types with higher specificity than previously reported.

scholarly journals Identification of ASCL1 as a determinant for human iPSC-derived dopaminergic neurons

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aaron M. Earley ◽  
Lena F. Burbulla ◽  
Dimitri Krainc ◽  
Rajeshwar Awatramani
Keyword(s):  
Stem Cells ◽  
Gene Regulation ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Fine Tuning ◽  
Transcriptional Networks ◽  
Single Cell Rna Sequencing

AbstractDuring cellular specification, transcription factors orchestrate cellular decisions through gene regulation. By hijacking these transcriptional networks, human pluripotent stem cells (hPSCs) can be specialized into neurons with different molecular identities for the purposes of regenerative medicine and disease modeling. However, molecular fine tuning cell types to match their in vivo counterparts remains a challenge. Directing cell fates often result in blended or incomplete neuron identities. A better understanding of hPSC to neuron gene regulation is needed. Here, we used single cell RNA sequencing to resolve some of these graded molecular identities during human neurogenesis from hPSCs. Differentiation platforms were established to model neural induction from stem cells, and we characterized these differentiated cell types by 10x single cell RNA sequencing. Using single cell trajectory and co-expression analyses, we identified a co-regulated transcription factor module expressing achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1) and neuronal differentiation 1 (NEUROD1). We then tested the function of these transcription factors in neuron subtype differentiation by gene knockout in a novel human system that reports the expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. ASCL1 was identified as a necessary transcription factor for regulating dopaminergic neurotransmitter selection.

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