RESUMEN
Non-neuronal cells are key to the complex cellular interplay that follows central nervous system insult. To understand this interplay, we generated a single-cell atlas of immune, glial and retinal pigment epithelial cells from adult mouse retina before and at multiple time points after axonal transection. We identified rare subsets in naive retina, including interferon (IFN)-response glia and border-associated macrophages, and delineated injury-induced changes in cell composition, expression programs and interactions. Computational analysis charted a three-phase multicellular inflammatory cascade after injury. In the early phase, retinal macroglia and microglia were reactivated, providing chemotactic signals concurrent with infiltration of CCR2+ monocytes from the circulation. These cells differentiated into macrophages in the intermediate phase, while an IFN-response program, likely driven by microglia-derived type I IFN, was activated across resident glia. The late phase indicated inflammatory resolution. Our findings provide a framework to decipher cellular circuitry, spatial relationships and molecular interactions following tissue injury.
Asunto(s)
Macrófagos , Retina , Animales , Ratones , Retina/lesiones , Retina/metabolismo , Microglía , Sistema Nervioso Central , MonocitosRESUMEN
Developing neurons connect in specific and stereotyped ways to form the complex circuits that underlie brain function. By comparison to earlier steps in neural development, progress has been slow in identifying the cell surface recognition molecules that mediate these synaptic choices, but new high-throughput imaging, genetic, and molecular methods are accelerating progress. Over the past decade, numerous large and small gene families have been implicated in target recognition, including members of the immunoglobulin, cadherin, and leucine-rich repeat superfamilies. We review these advances and propose ways in which combinatorial use of multifunctional recognition molecules enables the complex neuron-neuron interactions that underlie synaptic specificity.
Asunto(s)
Red Nerviosa/metabolismo , Red Nerviosa/fisiología , Sinapsis/metabolismo , Animales , Cadherinas/genética , Comunicación Celular , Membrana Celular/metabolismo , Humanos , Neurogénesis , Neuronas/metabolismo , Neuronas/fisiología , Sinapsis/fisiologíaRESUMEN
High-acuity vision in primates, including humans, is mediated by a small central retinal region called the fovea. As more accessible organisms lack a fovea, its specialized function and its dysfunction in ocular diseases remain poorly understood. We used 165,000 single-cell RNA-seq profiles to generate comprehensive cellular taxonomies of macaque fovea and peripheral retina. More than 80% of >60 cell types match between the two regions but exhibit substantial differences in proportions and gene expression, some of which we relate to functional differences. Comparison of macaque retinal types with those of mice reveals that interneuron types are tightly conserved. In contrast, projection neuron types and programs diverge, despite exhibiting conserved transcription factor codes. Key macaque types are conserved in humans, allowing mapping of cell-type and region-specific expression of >190 genes associated with 7 human retinal diseases. Our work provides a framework for comparative single-cell analysis across tissue regions and species.
Asunto(s)
Fóvea Central/fisiología , Primates/fisiología , Retina/fisiología , Anciano , Animales , Callithrix , Femenino , Humanos , Macaca , Masculino , Retina/anatomía & histología , Células Ganglionares de la Retina/metabolismoRESUMEN
The selectivity with which proprioceptive sensory neurons innervate their central and peripheral targets implies that they exhibit distinctions in muscle-type identity. The molecular correlates of proprioceptor identity and its origins remain largely unknown, however. In screens to define muscle-type proprioceptor character, we find all-or-none differences in gene expression for proprioceptors that control antagonistic muscles at a single hindlimb joint. Analysis of three of these genes, cadherin13 (cdh13), semaphorin5a (sema5a), and cartilage-acidic protein-1 (crtac1), reveals expression in proprioceptor subsets that supply muscle groups located at restricted dorsoventral and proximodistal domains of the limb. Genetically altering the dorsoventral character of the limb mesenchyme elicits a change in the profile of proprioceptor cdh13, sema5a, and crtac1 expression. These findings indicate that proprioceptors acquire aspects of their muscle-type identity in response to mesenchymal signals expressed in restricted proximodistal and dorsoventral domains of the developing limb.
Asunto(s)
Extremidades/embriología , Mesodermo/metabolismo , Propiocepción , Animales , Cadherinas/genética , Proteínas de Unión al Calcio/genética , Embrión de Mamíferos/metabolismo , Extremidades/fisiología , Ratones , Músculo Esquelético/inervación , Neuronas/metabolismo , Semaforinas/genética , Transducción de Señal , TranscriptomaRESUMEN
Although a number of repair strategies have been shown to promote axon outgrowth following neuronal injury in the mammalian CNS, it remains unclear whether regenerated axons establish functional synapses and support behavior. Here, in both juvenile and adult mice, we show that either PTEN and SOCS3 co-deletion, or co-overexpression of osteopontin (OPN)/insulin-like growth factor 1 (IGF1)/ciliary neurotrophic factor (CNTF), induces regrowth of retinal axons and formation of functional synapses in the superior colliculus (SC) but not significant recovery of visual function. Further analyses suggest that regenerated axons fail to conduct action potentials from the eye to the SC due to lack of myelination. Consistent with this idea, administration of voltage-gated potassium channel blockers restores conduction and results in increased visual acuity. Thus, enhancing both regeneration and conduction effectively improves function after retinal axon injury.
Asunto(s)
Axones/fisiología , Colículos Superiores/fisiología , 4-Aminopiridina/farmacología , Animales , Axones/efectos de los fármacos , Factor Neurotrófico Ciliar/metabolismo , Fenómenos Electrofisiológicos , Ojo/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Vaina de Mielina/metabolismo , Nervio Óptico , Osteopontina/metabolismo , Fosfohidrolasa PTEN/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Regeneración/efectos de los fármacos , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , SinapsisRESUMEN
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 â¼25,000 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.
Asunto(s)
Células Bipolares de la Retina/clasificación , Transcriptoma , Células Amacrinas/citología , Animales , Análisis por Conglomerados , Femenino , Marcadores Genéticos , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Células Bipolares de la Retina/citología , Células Bipolares de la Retina/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual/métodos , Transcripción GenéticaRESUMEN
Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Técnicas Analíticas Microfluídicas , Retina/citología , Análisis de la Célula Individual , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Análisis de Secuencia de ARNRESUMEN
Complex retinal circuits process visual information and deliver it to the brain. Few molecular determinants of synaptic specificity in this system are known. Using genetic and optogenetic methods, we identified two types of bipolar interneurons that convey visual input from photoreceptors to a circuit that computes the direction in which objects are moving. We then sought recognition molecules that promote selective connections of these cells with previously characterized components of the circuit. We found that the type II cadherins, cdh8 and cdh9, are each expressed selectively by one of the two bipolar cell types. Using loss- and gain-of-function methods, we showed that they are critical determinants of connectivity in this circuit and that perturbation of their expression leads to distinct defects in visually evoked responses. Our results reveal cellular components of a retinal circuit and demonstrate roles of type II cadherins in synaptic choice and circuit function.
Asunto(s)
Cadherinas/metabolismo , Retina/fisiología , Células Bipolares de la Retina/metabolismo , Vías Visuales , Animales , Axones/metabolismo , Cadherinas/genética , Técnicas de Sustitución del Gen , Ratones , Retina/citología , SinapsisRESUMEN
The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs1. 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 primates2. 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.
Asunto(s)
Evolución Biológica , Neuronas , Retina , Vertebrados , Visión Ocular , Animales , Humanos , Neuronas/clasificación , Neuronas/citología , Neuronas/fisiología , Retina/citología , Retina/fisiología , Células Ganglionares de la Retina/clasificación , Análisis de Expresión Génica de una Sola Célula , Vertebrados/fisiología , Visión Ocular/fisiología , Especificidad de la Especie , Células Amacrinas/clasificación , Células Fotorreceptoras/clasificación , Células Ependimogliales/clasificación , Células Bipolares de la Retina/clasificación , Percepción VisualRESUMEN
The nervous system is populated by numerous types of neurons, each bearing a dendritic arbor with a characteristic morphology. These type-specific features influence many aspects of a neuron's function, including the number and identity of presynaptic inputs and how inputs are integrated to determine firing properties. Here, we review the mechanisms that regulate the construction of cell type-specific dendrite patterns during development. We focus on four aspects of dendrite patterning that are particularly important in determining the function of the mature neuron: (a) dendrite shape, including branching pattern and geometry of the arbor; (b) dendritic arbor size;
Asunto(s)
Dendritas/fisiología , Animales , Emparejamiento Cromosómico/fisiología , HumanosRESUMEN
In primates, high-acuity vision is mediated by the fovea, a small specialized central region of the retina. The fovea, unique to the anthropoid lineage among mammals, undergoes notable neuronal morphological changes during postnatal maturation. However, the extent of cellular similarity across anthropoid foveas and the molecular underpinnings of foveal maturation remain unclear. Here, we used high-throughput single-cell RNA sequencing to profile retinal cells of the common marmoset (Callithrix jacchus), an early divergent in anthropoid evolution from humans, apes, and macaques. We generated atlases of the marmoset fovea and peripheral retina for both neonates and adults. Our comparative analysis revealed that marmosets share almost all their foveal types with both humans and macaques, highlighting a conserved cellular structure among primate foveas. Furthermore, by tracing the developmental trajectory of cell types in the foveal and peripheral retina, we found distinct maturation paths for each. In-depth analysis of gene expression differences demonstrated that cone photoreceptors and Müller glia (MG), among others, show the greatest molecular divergence between these two regions. Utilizing single-cell ATAC-seq and gene-regulatory network inference, we uncovered distinct transcriptional regulations differentiating foveal cones from their peripheral counterparts. Further analysis of predicted ligand-receptor interactions suggested a potential role for MG in supporting the maturation of foveal cones. Together, these results provide valuable insights into foveal development, structure, and evolution.
Asunto(s)
Callithrix , Retina , Humanos , Animales , Recién Nacido , Callithrix/anatomía & histología , Retina/metabolismo , Fóvea Central/fisiología , Células Fotorreceptoras Retinianas Conos , Macaca , MamíferosRESUMEN
Although the visual system extends through the brain, most vision loss originates from defects in the eye. Its central element is the neural retina, which senses light, processes visual signals, and transmits them to the rest of the brain through the optic nerve (ON). Surrounding the retina are numerous other structures, conventionally divided into anterior and posterior segments. Here, we used high-throughput single-nucleus RNA sequencing (snRNA-seq) to classify and characterize cells in six extraretinal components of the posterior segment: ON, optic nerve head (ONH), peripheral sclera, peripapillary sclera (PPS), choroid, and retinal pigment epithelium (RPE). Defects in each of these tissues are associated with blinding diseases-for example, glaucoma (ONH and PPS), optic neuritis (ON), retinitis pigmentosa (RPE), and age-related macular degeneration (RPE and choroid). From ~151,000 single nuclei, we identified 37 transcriptomically distinct cell types, including multiple types of astrocytes, oligodendrocytes, fibroblasts, and vascular endothelial cells. Our analyses revealed a differential distribution of many cell types among distinct structures. Together with our previous analyses of the anterior segment and retina, the data presented here complete a "Version 1" cell atlas of the human eye. We used this atlas to map the expression of >180 genes associated with the risk of developing glaucoma, which is known to involve ocular tissues in both anterior and posterior segments as well as the neural retina. Similar methods can be used to investigate numerous additional ocular diseases, many of which are currently untreatable.
Asunto(s)
Glaucoma , Disco Óptico , Humanos , Transcriptoma , Células Endoteliales , Glaucoma/genética , Nervio Óptico , EscleróticaRESUMEN
The chemoaffinity hypothesis for neural circuit assembly posits that axons and their targets bear matching molecular labels that endow neurons with unique identities and specify synapses between appropriate partners. Here, we focus on two intriguing candidates for fulfilling this role, Drosophila Dscams and vertebrate clustered protocadherins (Pcdhs). In each, a complex genomic locus encodes large numbers of neuronal transmembrane proteins with homophilic binding specificity, individual members of which are expressed combinatorially. Although these properties suggest that Dscams and Pcdhs could act as specificity molecules, they may do so in ways that challenge traditional views of how neural circuits assemble.
Asunto(s)
Cadherinas/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Drosophila/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis , Neuronas/metabolismo , Animales , Axones/metabolismo , Dendritas/metabolismo , Drosophila/embriología , Neuronas/citología , Vertebrados/embriologíaRESUMEN
The anterior segment of the eye consists of the cornea, iris, ciliary body, crystalline lens, and aqueous humor outflow pathways. Together, these tissues are essential for the proper functioning of the eye. Disorders of vision have been ascribed to defects in all of them; some disorders, including glaucoma and cataract, are among the most prevalent causes of blindness in the world. To characterize the cell types that compose these tissues, we generated an anterior segment cell atlas of the human eye using high-throughput single-nucleus RNA sequencing (snRNAseq). We profiled 195,248 nuclei from nondiseased anterior segment tissues of six human donors, identifying >60 cell types. Many of these cell types were discrete, whereas others, especially in the lens and cornea, formed continua corresponding to known developmental transitions that persist in adulthood. Having profiled each tissue separately, we performed an integrated analysis of the entire anterior segment, revealing that some cell types are unique to a single structure, whereas others are shared across tissues. The integrated cell atlas was then used to investigate cell type-specific expression patterns of more than 900 human ocular disease genes identified through either Mendelian inheritance patterns or genome-wide association studies.
Asunto(s)
Segmento Anterior del Ojo , Oftalmopatías , Adulto , Segmento Anterior del Ojo/citología , Segmento Anterior del Ojo/metabolismo , Humor Acuoso/citología , Humor Acuoso/metabolismo , Atlas como Asunto , Cuerpo Ciliar/citología , Cuerpo Ciliar/metabolismo , Oftalmopatías/genética , Estudio de Asociación del Genoma Completo , Humanos , Iris/citología , Especificidad de ÓrganosRESUMEN
In the retina, photoreceptors pass visual information to interneurons, which process it and pass it to retinal ganglion cells (RGCs). Axons of RGCs then travel through the optic nerve, telling the rest of the brain all it will ever know about the visual world. Research over the past several decades has made clear that most RGCs are not merely light detectors, but rather feature detectors, which send a diverse set of parallel, highly processed images of the world on to higher centers. Here, we review progress in classification of RGCs by physiological, morphological, and molecular criteria, making a particular effort to distinguish those cell types that are definitive from those for which information is partial. We focus on the mouse, in which molecular and genetic methods are most advanced. We argue that there are around 30 RGC types and that we can now account for well over half of all RGCs. We also use RGCs to examine the general problem of neuronal classification, arguing that insights and methods from the retina can guide the classification enterprise in other brain regions.
Asunto(s)
Células Ganglionares de la Retina/clasificación , Animales , Evolución Biológica , Modelos NeurológicosRESUMEN
The most impressive structural feature of the nervous system is the specificity of its synaptic connections. Even after axons have navigated long distances to reach target areas, they must still choose appropriate synaptic partners from the many potential partners within easy reach. In many cases, axons also select a particular domain of the postsynaptic cell on which to form a synapse. Thus, synapse formation is selective at both cellular and subcellular levels. Unsurprisingly, the nervous system uses multiple mechanisms to ensure proper connectivity; these include complementary labels, coordinated growth of synaptic partners, sorting of afferents, prohibition or elimination of inappropriate synapses, respecification of targets, and use of short-range guidance mechanisms or intermediate targets. Specification of any circuit is likely to involve integration of multiple mechanisms. Recent studies of vertebrate and invertebrate systems have led to the identification of molecules that mediate a few of these interactions.
Asunto(s)
Sistema Nervioso/metabolismo , Sinapsis/metabolismo , Animales , Axones/metabolismo , Dendritas/metabolismo , Modelos Neurológicos , Neuronas/metabolismo , Transmisión SinápticaRESUMEN
Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From â¼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa), and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions.
Asunto(s)
Humor Acuoso/metabolismo , Modelos Animales de Enfermedad , Ojo/metabolismo , Glaucoma/patología , Presión Intraocular , Malla Trabecular/metabolismo , Transcriptoma , Animales , Biomarcadores/análisis , Ojo/citología , Glaucoma/metabolismo , Humanos , Macaca fascicularis , Macaca mulatta , Ratones , Especificidad de la Especie , PorcinosRESUMEN
Neurons have diverse molecular, morphological, connectional and functional properties. We believe that the only realistic way to manage this complexity - and thereby pave the way for understanding the structure, function and development of brain circuits - is to group neurons into types, which can then be analysed systematically and reproducibly. However, neuronal classification has been challenging both technically and conceptually. New high-throughput methods have created opportunities to address the technical challenges associated with neuronal classification by collecting comprehensive information about individual cells. Nonetheless, conceptual difficulties persist. Borrowing from the field of species taxonomy, we propose principles to be followed in the cell-type classification effort, including the incorporation of multiple, quantitative features as criteria, the use of discontinuous variation to define types and the creation of a hierarchical system to represent relationships between cells. We review the progress of classifying cell types in the retina and cerebral cortex and propose a staged approach for moving forward with a systematic cell-type classification in the nervous system.
Asunto(s)
Corteza Cerebral/citología , Neuronas/clasificación , Neuronas/fisiología , Retina/citología , Animales , HumanosRESUMEN
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 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 morphologic analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in the mouse retina.SIGNIFICANCE STATEMENT 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 â¼130 neuronal types and is therefore comparable in complexity to other regions of the brain.