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Nature Communications Oct 2019Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in...
Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. However, it has been challenging to identify the cell types associated with AMD given the genetic complexity of the disease. Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the first single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Heterogeneity is observed within macroglia, suggesting that human retinal glia are more diverse than previously thought. Finally, GWAS-based enrichment analysis identifies glia, vascular cells, and cone photoreceptors to be associated with the risk of AMD. These data provide a detailed analysis of the human retina, and show how scRNA-seq can provide insight into cell types involved in complex, inflammatory genetic diseases.
Topics: Amacrine Cells; Astrocytes; Blood Vessels; Ependymoglial Cells; Gene Expression; Gene Expression Profiling; Genetic Predisposition to Disease; High-Throughput Nucleotide Sequencing; Humans; Macular Degeneration; Microglia; Neuroglia; Retina; Retinal Bipolar Cells; Retinal Cone Photoreceptor Cells; Retinal Ganglion Cells; Retinal Horizontal Cells; Retinal Neurons; Retinal Rod Photoreceptor Cells; Retinal Vessels; Sequence Analysis, RNA; Single-Cell Analysis
PubMed: 31653841
DOI: 10.1038/s41467-019-12780-8 -
The Journal of Neuroscience : the... Jul 2020Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively...
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 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 expressed 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 and , 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 morphologic analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in the mouse retina. The 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 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 the 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.
Topics: Amacrine Cells; Animals; Female; Glycine; High-Throughput Nucleotide Sequencing; Homeodomain Proteins; Male; Mice; Mice, Inbred C57BL; Neuropeptides; Neurotransmitter Agents; Receptors, Neurotransmitter; Retina; Transcription Factor 4; Transcription Factors; gamma-Aminobutyric Acid
PubMed: 32457074
DOI: 10.1523/JNEUROSCI.0471-20.2020 -
Proceedings of the National Academy of... Nov 2023Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to...
Diabetic retinopathy (DR) is a neurovascular complication of diabetes. Recent investigations have suggested that early degeneration of the neuroretina may occur prior to the appearance of microvascular changes; however, the mechanisms underlying this neurodegeneration have been elusive. Microglia are the predominant resident immune cell in the retina and adopt dynamic roles in disease. Here, we show that ablation of retinal microglia ameliorates visual dysfunction and neurodegeneration in a type I diabetes mouse model. We also provide evidence of enhanced microglial contact and engulfment of amacrine cells, ultrastructural modifications, and transcriptome changes that drive inflammation and phagocytosis. We show that CD200-CD200R signaling between amacrine cells and microglia is dysregulated during early DR and that targeting CD200R can attenuate high glucose-induced inflammation and phagocytosis in cultured microglia. Last, we demonstrate that targeting CD200R in vivo can prevent visual dysfunction, microglia activation, and retinal inflammation in the diabetic mouse. These studies provide a molecular framework for the pivotal role that microglia play in early DR pathogenesis and identify a potential immunotherapeutic target for treating DR in patients.
Topics: Animals; Humans; Mice; Diabetes Mellitus; Diabetic Retinopathy; Inflammation; Microglia; Retina; Signal Transduction
PubMed: 37903272
DOI: 10.1073/pnas.2308214120 -
Cell Aug 2016Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential...
Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.
Topics: Amacrine Cells; Animals; Cluster Analysis; Female; Genetic Markers; Male; Mice; Mice, Inbred Strains; Mice, Transgenic; Retinal Bipolar Cells; Sequence Analysis, RNA; Single-Cell Analysis; Transcription, Genetic; Transcriptome
PubMed: 27565351
DOI: 10.1016/j.cell.2016.07.054 -
Nature Communications Aug 2023The visual signal processing in the retina requires the precise organization of diverse neuronal types working in concert. While single-cell omics studies have...
The visual signal processing in the retina requires the precise organization of diverse neuronal types working in concert. While single-cell omics studies have identified more than 120 different neuronal subtypes in the mouse retina, little is known about their spatial organization. Here, we generated the single-cell spatial atlas of the mouse retina using multiplexed error-robust fluorescence in situ hybridization (MERFISH). We profiled over 390,000 cells and identified all major cell types and nearly all subtypes through the integration with reference single-cell RNA sequencing (scRNA-seq) data. Our spatial atlas allowed simultaneous examination of nearly all cell subtypes in the retina, revealing 8 previously unknown displaced amacrine cell subtypes and establishing the connection between the molecular classification of many cell subtypes and their spatial arrangement. Furthermore, we identified spatially dependent differential gene expression between subtypes, suggesting the possibility of functional tuning of neuronal types based on location.
Topics: Animals; Mice; Gene Expression Profiling; In Situ Hybridization, Fluorescence; Retina; Amacrine Cells; Single-Cell Analysis
PubMed: 37582959
DOI: 10.1038/s41467-023-40674-3 -
Nature Dec 2023The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs. Retinal cell types may have evolved to accommodate... (Comparative Study)
Comparative Study
The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs. Retinal cell types may have evolved to accommodate these varied needs, but this has not been systematically studied. Here we generated and integrated single-cell transcriptomic atlases of the retina from 17 species: humans, two non-human primates, four rodents, three ungulates, opossum, ferret, tree shrew, a bird, a reptile, a teleost fish and a lamprey. We found high molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells (RGCs) and Müller glia), with transcriptomic variation across species related to evolutionary distance. Major subclasses were also conserved, whereas variation among cell types within classes or subclasses was more pronounced. However, an integrative analysis revealed that numerous cell types are shared across species, based on conserved gene expression programmes that are likely to trace back to an early ancestral vertebrate. The degree of variation among cell types increased from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified rodent orthologues of midget RGCs, which comprise more than 80% of RGCs in the human retina, subserve high-acuity vision, and were previously believed to be restricted to primates. By contrast, the mouse orthologues have large receptive fields and comprise around 2% of mouse RGCs. Projections of both primate and mouse orthologous types are overrepresented in the thalamus, which supplies the primary visual cortex. We suggest that midget RGCs are not primate innovations, but are descendants of evolutionarily ancient types that decreased in size and increased in number as primates evolved, thereby facilitating high visual acuity and increased cortical processing of visual information.
Topics: Animals; Humans; Neurons; Retina; Retinal Ganglion Cells; Single-Cell Gene Expression Analysis; Vertebrates; Vision, Ocular; Species Specificity; Biological Evolution; Amacrine Cells; Photoreceptor Cells; Ependymoglial Cells; Retinal Bipolar Cells; Visual Perception
PubMed: 38092908
DOI: 10.1038/s41586-023-06638-9 -
Visual Neuroscience Jan 2016Amacrine cells are a diverse set of local circuit neurons of the inner retina, and they all release either GABA or glycine, amino acid neurotransmitters that are... (Review)
Review
Amacrine cells are a diverse set of local circuit neurons of the inner retina, and they all release either GABA or glycine, amino acid neurotransmitters that are generally inhibitory. But some types of amacrine cells have another function besides inhibiting other neurons. One glycinergic amacrine cell, the Aii type, excites a subset of bipolar cells via extensive gap junctions while inhibiting others at chemical synapses. Many types of GABAergic amacrine cells also release monoamines, acetylcholine, or neuropeptides. There is now good evidence that another type of amacrine cell releases glycine at some of its synapses and releases the excitatory amino acid glutamate at others. The glutamatergic synapses are made onto a subset of retinal ganglion cells and amacrine cells and have the asymmetric postsynaptic densities characteristic of central excitatory synapses. The glycinergic synapses are made onto other types of ganglion cells and have the symmetric postsynaptic densities characteristic of central inhibitory synapses. These amacrine cells, which contain vesicular glutamate transporter 3, will be the focus of this brief review.
Topics: Amacrine Cells; Animals; GABA Plasma Membrane Transport Proteins; Glucose Transporter Type 3; Glycine Plasma Membrane Transport Proteins; Humans
PubMed: 28359349
DOI: 10.1017/S0952523816000146 -
Progress in Retinal and Eye Research Feb 2020This review summarizes our current knowledge of primate including human retina focusing on bipolar, amacrine and ganglion cells and their connectivity. We have two main... (Review)
Review
This review summarizes our current knowledge of primate including human retina focusing on bipolar, amacrine and ganglion cells and their connectivity. We have two main motivations in writing. Firstly, recent progress in non-invasive imaging methods to study retinal diseases mean that better understanding of the primate retina is becoming an important goal both for basic and for clinical sciences. Secondly, genetically modified mice are increasingly used as animal models for human retinal diseases. Thus, it is important to understand to which extent the retinas of primates and rodents are comparable. We first compare cell populations in primate and rodent retinas, with emphasis on how the fovea (despite its small size) dominates the neural landscape of primate retina. We next summarise what is known, and what is not known, about the postreceptoral neurone populations in primate retina. The inventories of bipolar and ganglion cells in primates are now nearing completion, comprising ~12 types of bipolar cell and at least 17 types of ganglion cell. Primate ganglion cells show clear differences in dendritic field size across the retina, and their morphology differs clearly from that of mouse retinal ganglion cells. Compared to bipolar and ganglion cells, amacrine cells show even higher morphological diversity: they could comprise over 40 types. Many amacrine types appear conserved between primates and mice, but functions of only a few types are understood in any primate or non-primate retina. Amacrine cells appear as the final frontier for retinal research in monkeys and mice alike.
PubMed: 32032773
DOI: 10.1016/j.preteyeres.2020.100844 -
Development (Cambridge, England) Jan 2022The mammalian retina contains a complex mixture of different types of neurons. We find that microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine...
The mammalian retina contains a complex mixture of different types of neurons. We find that 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 retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b directed the formation of additional amacrine cells and reduced bipolar neurons in the developing retina. We identify the Foxn3 mRNA as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3, a transcription factor, in the postnatal developing retina by RNAi increased the formation of amacrine cells and reduced bipolar cell formation. Foxn3 disruption by CRISPR in embryonic retinal explants also increased amacrine cell formation, whereas Foxn3 overexpression inhibited amacrine cell formation prior to Ptf1a expression. Co-expression of Foxn3 partially reversed the effects of ectopic miR-216b on retinal cell formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates Foxn3 and other genes in amacrine cells.
Topics: Amacrine Cells; Animals; Cell Cycle Proteins; Female; Forkhead Transcription Factors; HEK293 Cells; Humans; Male; Mice; MicroRNAs; Neurogenesis; Transcription Factors
PubMed: 34919141
DOI: 10.1242/dev.199484