Evolution of neuronal cell classes and types in the vertebrate retina.

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.

Integrated multi-omics single cell atlas of the human retina.

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here, we present an integrated multimodal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 2.4 million cells from 55 donors, including 1.4 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) of transcriptome and chromatin accessibility, unveiling over 110 types. Engaging the retina community, we annotated each cluster, refined the Cell Ontology for the retina, identified distinct marker genes, and characterized cis-regulatory elements and gene regulatory networks (GRNs) for these cell types. Our analysis uncovered intriguing differences in transcriptome, chromatin, and GRNs across cell types. In addition, we modeled changes in gene expression and chromatin openness across gender and age. This integrated atlas also enabled the fine-mapping of GWAS and eQTL variants. Accessible through interactive browsers, this multimodal cross-donor and cross-lab HRCA, can facilitate a better understanding of retinal function and pathology.

Suppressing DNMT3a Alleviates the Intrinsic Epigenetic Barrier for Optic Nerve Regeneration and Restores Vision in Adult Mice.

The limited regenerative potential of the optic nerve in adult mammals presents a major challenge for restoring vision after optic nerve trauma or disease. The mechanisms of this regenerative failure are not fully understood1,2. Here, through small-molecule and genetic screening for epigenetic modulators3, we identify DNA methyltransferase 3a (DNMT3a) as a potent inhibitor of axon regeneration in mouse and human retinal explants. Selective suppression of DNMT3a in retinal ganglion cells (RGCs) by gene targeting or delivery of shRNA leads to robust, full-length regeneration of RGC axons through the optic nerve and restoration of vision in adult mice after nerve crush injury. Genome-wide bisulfite and transcriptome profiling in combination with single nucleus RNA-sequencing of RGCs revealed selective DNA demethylation and reactivation of genetic programs supporting neuronal survival and axonal growth/regeneration by DNMT3a deficiency. This was accompanied by the suppression of gene networks associated with apoptosis and inflammation. Our results identify DNMT3a as the central orchestrator of an RGC-intrinsic mechanism that limits optic nerve regeneration. Suppressing DNMT3a expression in RGCs unlocks the epigenetic switch for optic nerve regeneration and presents a promising therapeutic avenue for effectively reversing vision loss resulted from optic nerve trauma or diseases.

Retinal ganglion cell repopulation for vision restoration in optic neuropathy: a roadmap from the RReSTORe Consortium.

Retinal ganglion cell (RGC) death in glaucoma and other optic neuropathies results in irreversible vision loss due to the mammalian central nervous system's limited regenerative capacity. RGC repopulation is a promising therapeutic approach to reverse vision loss from optic neuropathies if the newly introduced neurons can reestablish functional retinal and thalamic circuits. In theory, RGCs might be repopulated through the transplantation of stem cell-derived neurons or via the induction of endogenous transdifferentiation. The RGC Repopulation, Stem Cell Transplantation, and Optic Nerve Regeneration (RReSTORe) Consortium was established to address the challenges associated with the therapeutic repair of the visual pathway in optic neuropathy. In 2022, the RReSTORe Consortium initiated ongoing international collaborative discussions to advance the RGC repopulation field and has identified five critical areas of focus: (1) RGC development and differentiation, (2) Transplantation methods and models, (3) RGC survival, maturation, and host interactions, (4) Inner retinal wiring, and (5) Eye-to-brain connectivity. Here, we discuss the most pertinent questions and challenges that exist on the path to clinical translation and suggest experimental directions to propel this work going forward. Using these five subtopic discussion groups (SDGs) as a framework, we suggest multidisciplinary approaches to restore the diseased visual pathway by leveraging groundbreaking insights from developmental neuroscience, stem cell biology, molecular biology, optical imaging, animal models of optic neuropathy, immunology & immunotolerance, neuropathology & neuroprotection, materials science & biomedical engineering, and regenerative neuroscience. While significant hurdles remain, the RReSTORe Consortium's efforts provide a comprehensive roadmap for advancing the RGC repopulation field and hold potential for transformative progress in restoring vision in patients suffering from optic neuropathies.

Transcriptomic analysis of the ocular posterior segment completes a cell atlas of the human eye.

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.

Artificial Intelligence Models for Cell Type and Subtype Identification Based on Single-Cell RNA Sequencing Data in Vision Science.

Single-cell RNA sequencing (scRNA-seq) provides a high throughput, quantitative and unbiased framework for scientists in many research fields to identify and characterize cell types within heterogeneous cell populations from various tissues. However, scRNA-seq based identification of discrete cell-types is still labor intensive and depends on prior molecular knowledge. Artificial intelligence has provided faster, more accurate, and user-friendly approaches for cell-type identification. In this review, we discuss recent advances in cell-type identification methods using artificial intelligence techniques based on single-cell and single-nucleus RNA sequencing data in vision science. The main purpose of this review paper is to assist vision scientists not only to select suitable datasets for their problems, but also to be aware of the appropriate computational tools to perform their analysis. Developing novel methods for scRNA-seq data analysis remains to be addressed in future studies.

Transcriptomic Analysis of the Ocular Posterior Segment Completes a Cell Atlas of the Human Eye.

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 the 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 new data 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 neural retina. Similar methods can be used to investigate numerous additional ocular diseases, many of which are currently untreatable.

Evolution of neuronal cell classes and types in the vertebrate retina.

The basic plan of the retina is conserved across vertebrates, yet species differ profoundly in their visual needs (Baden et al., 2020). One might expect that retinal cell types 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 teleost fish, a bird, a reptile and a lamprey. Molecular conservation of the six retinal cell classes (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells [RGCs] and Muller glia) is striking, with transcriptomic differences across species correlated with evolutionary distance. Major subclasses are also conserved, whereas variation among types within classes or subclasses is more pronounced. However, an integrative analysis revealed that numerous types are shared across species based on conserved gene expression programs that likely trace back to the common ancestor of jawed vertebrates. The degree of variation among types increases from the outer retina (photoreceptors) to the inner retina (RGCs), suggesting that evolution acts preferentially to shape the retinal output. Finally, we identified mammalian orthologs of midget RGCs, which comprise >80% of RGCs in the human retina, subserve high-acuity vision, and were believed to be primate-specific (Berson, 2008); in contrast, the mouse orthologs comprise <2% of mouse RGCs. Projections 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 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.

Cell atlas of the human ocular anterior segment: Tissue-specific and shared cell types.

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.

Core transcription programs controlling injury-induced neurodegeneration of retinal ganglion cells.

Regulatory programs governing neuronal death and axon regeneration in neurodegenerative diseases remain poorly understood. In adult mice, optic nerve crush (ONC) injury by severing retinal ganglion cell (RGC) axons results in massive RGC death and regenerative failure. We performed an in vivo CRISPR-Cas9-based genome-wide screen of 1,893 transcription factors (TFs) to seek repressors of RGC survival and axon regeneration following ONC. In parallel, we profiled the epigenetic and transcriptional landscapes of injured RGCs by ATAC-seq and RNA-seq to identify injury-responsive TFs and their targets. These analyses converged on four TFs as critical survival regulators, of which ATF3/CHOP preferentially regulate pathways activated by cytokines and innate immunity and ATF4/C/EBPγ regulate pathways engaged by intrinsic neuronal stressors. Manipulation of these TFs protects RGCs in a glaucoma model. Our results reveal core transcription programs that transform an initial axonal insult into a degenerative process and suggest novel strategies for treating neurodegenerative diseases.

Pericyte heterogeneity identified by 3D ultrastructural analysis of the microvessel wall.

Confident identification of pericytes (PCs) remains an obstacle in the field, as a single molecular marker for these unique perivascular cells remains elusive. Adding to this challenge is the recent appreciation that PC populations may be heterogeneous, displaying a range of morphologies within capillary networks. We found additional support on the ultrastructural level for the classification of these PC subtypes-"thin-strand" (TSP), mesh (MP), and ensheathing (EP)-based on distinct morphological characteristics. Interestingly, we also found several examples of another cell type, likely a vascular smooth muscle cell, in a medial layer between endothelial cells (ECs) and pericytes (PCs) harboring characteristics of the ensheathing type. A conserved feature across the different PC subtypes was the presence of extracellular matrix (ECM) surrounding the vascular unit and distributed in between neighboring cells. The thickness of this vascular basement membrane was remarkably consistent depending on its location, but never strayed beyond a range of 150-300 nm unless thinned to facilitate closer proximity of neighboring cells (suggesting direct contact). The density of PC-EC contact points ("peg-and-socket" structures) was another distinguishing feature across the different PC subtypes, as were the apparent contact locations between vascular cells and brain parenchymal cells. In addition to this thinning, the extracellular matrix (ECM) surrounding EPs displayed another unique configuration in the form of extensions that emitted out radially into the surrounding parenchyma. Knowledge of the origin and function of these structures is still emerging, but their appearance suggests the potential for being mechanical elements and/or perhaps signaling nodes via embedded molecular cues. Overall, this unique ultrastructural perspective provides new insights into PC heterogeneity and the presence of medial cells within the microvessel wall, the consideration of extracellular matrix (ECM) coverage as another PC identification criteria, and unique extracellular matrix (ECM) configurations (i.e., radial extensions) that may reveal additional aspects of PC heterogeneity.

Microglia-organized scar-free spinal cord repair in neonatal mice.

Spinal cord injury in mammals is thought to trigger scar formation with little regeneration of axons1-4. Here we show that a crush injury to the spinal cord in neonatal mice leads to scar-free healing that permits the growth of long projecting axons through the lesion. Depletion of microglia in neonatal mice disrupts this healing process and stalls the regrowth of axons, suggesting that microglia are critical for orchestrating the injury response. Using single-cell RNA sequencing and functional analyses, we find that neonatal microglia are transiently activated and have at least two key roles in scar-free healing. First, they transiently secrete fibronectin and its binding proteins to form bridges of extracellular matrix that ligate the severed ends of the spinal cord. Second, neonatal-but not adult-microglia express several extracellular and intracellular peptidase inhibitors, as well as other molecules that are involved in resolving inflammation. We transplanted either neonatal microglia or adult microglia treated with peptidase inhibitors into spinal cord lesions of adult mice, and found that both types of microglia significantly improved healing and axon regrowth. Together, our results reveal the cellular and molecular basis of the nearly complete recovery of neonatal mice after spinal cord injury, and suggest strategies that could be used to facilitate scar-free healing in the adult mammalian nervous system.

Retinal inputs signal astrocytes to recruit interneurons into visual thalamus.

Inhibitory interneurons comprise a fraction of the total neurons in the visual thalamus but are essential for sharpening receptive field properties and improving contrast-gain of retinogeniculate transmission. During early development, these interneurons undergo long-range migration from germinal zones, a process regulated by the innervation of the visual thalamus by retinal ganglion cells. Here, using transcriptomic approaches, we identified a motogenic cue, fibroblast growth factor 15 (FGF15), whose expression in the visual thalamus is regulated by retinal input. Targeted deletion of functional FGF15 in mice led to a reduction in thalamic GABAergic interneurons similar to that observed in the absence of retinal input. This loss may be attributed, at least in part, to misrouting of interneurons into nonvisual thalamic nuclei. Unexpectedly, expression analysis revealed that FGF15 is generated by thalamic astrocytes and not retino-recipient neurons. Thus, these data show that retinal inputs signal through astrocytes to direct the long-range recruitment of interneurons into the visual thalamus.

Retinal-input-induced epigenetic dynamics in the developing mouse dorsal lateral geniculate nucleus.

DNA methylation plays important roles in the regulation of nervous system development and in cellular responses to environmental stimuli such as light-derived signals. Despite great efforts in understanding the maturation and refinement of visual circuits, we lack a clear understanding of how changes in DNA methylation correlate with visual activity in the developing subcortical visual system, such as in the dorsal lateral geniculate nucleus (dLGN), the main retino-recipient region in the dorsal thalamus. Here, we explored epigenetic dynamics underlying dLGN development at ages before and after eye opening in wild-type mice and mutant mice in which retinal ganglion cells fail to form. We observed that development-related epigenetic changes tend to co-localize together on functional genomic regions critical for regulating gene expression, while retinal-input-induced epigenetic changes are enriched on repetitive elements. Enhancers identified in neurons are prone to methylation dynamics during development, and activity-induced enhancers are associated with retinal-input-induced epigenetic changes. Intriguingly, the binding motifs of activity-dependent transcription factors, including EGR1 and members of MEF2 family, are enriched in the genomic regions with epigenetic aberrations in dLGN tissues of mutant mice lacking retinal inputs. Overall, our study sheds new light on the epigenetic regulatory mechanisms underlying the role of retinal inputs on the development of mouse dLGN.

Distribution and development of molecularly distinct perineuronal nets in visual thalamus.

Visual information is detected by the retina and transmitted into the brain by retinal ganglion cells. In rodents, the visual thalamus is a major recipient of retinal ganglion cells axons and is divided into three functionally distinct nuclei: the dorsal lateral geniculate nucleus (dLGN), ventral LGN (vLGN), and intergeniculate leaflet. Despite being densely innervated by retinal input, each nucleus in rodent visual thalamus possesses diverse molecular profiles which underpin their unique circuitry and cytoarchitecture. Here, we combined large-scale unbiased proteomic and transcriptomic analyses to elucidate the molecular expression profiles of the developing mouse dLGN and vLGN. We identified several extracellular matrix proteins as differentially expressed in these regions, particularly constituent molecules of perineuronal nets (PNNs). Remarkably, we discovered at least two types of molecularly distinct Aggrecan-rich PNN populations in vLGN, exhibiting non-overlapping spatial, temporal, and cell-type specific expression patterns. The mechanisms responsible for the formation of these two populations of PNNs also differ as the formation of Cat315+ PNNs (but not WFA+ PNNs) required input from the retina. This study is first to suggest that cell type- and molecularly specific supramolecular assemblies of extracellular matrix may play important roles in the circuitry associated with the subcortical visual system and in the processing of visual information. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Cover Image for this issue: doi: 10.1111/jnc.14203.

F-spondin Is Essential for Maintaining Circadian Rhythms.

The suprachiasmatic nucleus (SCN) is the master pacemaker that drives circadian behaviors. SCN neurons have intrinsic, self-sustained rhythmicity that is governed by transcription-translation feedback loops. Intrinsic rhythms within the SCN do not match the day-night cycle and are therefore entrained by light-derived cues. Such cues are transmitted to the SCN by a class of intrinsically photosensitive retinal ganglion cells (ipRGCs). In the present study, we sought to identify how axons from ipRGCs target the SCN. While none of the potential targeting cues identified appeared necessary for retinohypothalamic innervation, we unexpectedly identified a novel role for the extracellular matrix protein F-spondin in circadian behavior. In the absence of F-spondin, mice lost their ability to maintain typical intrinsic rhythmicity. Moreover, F-spondin loss results in the displacement of vasoactive intestinal peptide (VIP)-expressing neurons, a class of neurons that are essential for maintaining rhythmicity among SCN neurons. Thus, this study highlights a novel role for F-spondin in maintaining circadian rhythms.

LRRTM1 underlies synaptic convergence in visual thalamus.

It has long been thought that the mammalian visual system is organized into parallel pathways, with incoming visual signals being parsed in the retina based on feature (e.g. color, contrast and motion) and then transmitted to the brain in unmixed, feature-specific channels. To faithfully convey feature-specific information from retina to cortex, thalamic relay cells must receive inputs from only a small number of functionally similar retinal ganglion cells. However, recent studies challenged this by revealing substantial levels of retinal convergence onto relay cells. Here, we sought to identify mechanisms responsible for the assembly of such convergence. Using an unbiased transcriptomics approach and targeted mutant mice, we discovered a critical role for the synaptic adhesion molecule Leucine Rich Repeat Transmembrane Neuronal 1 (LRRTM1) in the emergence of retinothalamic convergence. Importantly, LRRTM1 mutant mice display impairment in visual behaviors, suggesting a functional role of retinothalamic convergence in vision.

Not a one-trick pony: Diverse connectivity and functions of the rodent lateral geniculate complex.

Often mislabeled as a simple relay of sensory information, the thalamus is a complicated structure with diverse functions. This diversity is exemplified by roles visual thalamus plays in processing and transmitting light-derived stimuli. Such light-derived signals are transmitted to the thalamus by retinal ganglion cells (RGCs), the sole projection neurons of the retina. Axons from RGCs innervate more than ten distinct nuclei within thalamus, including those of the lateral geniculate complex. Nuclei within the lateral geniculate complex of nocturnal rodents, which include the dorsal lateral geniculate nucleus (dLGN), ventral lateral geniculate nucleus (vLGN), and intergeniculate leaflet (IGL), are each densely innervated by retinal projections, yet, exhibit distinct cytoarchitecture and connectivity. These features suggest that each nucleus within this complex plays a unique role in processing and transmitting light-derived signals. Here, we review the diverse cytoarchitecture and connectivity of these nuclei in nocturnal rodents, in an effort to highlight roles for dLGN in vision and for vLGN and IGL in visuomotor, vestibular, ocular, and circadian function.

Region- and Cell-Specific Expression of Transmembrane Collagens in Mouse Brain.

Unconventional collagens are nonfribrillar proteins that not only contribute to the structure of extracellular matrices but exhibit unique bio-activities. Although roles for unconventional collagens have been well-established in the development and function of non-neural tissues, only recently have studies identified roles for these proteins in brain development, and more specifically, in the formation and refinement of synaptic connections between neurons. Still, our understanding of the full cohort of unconventional collagens that are generated in the mammalian brain remains unclear. Here, we sought to address this gap by assessing the expression of transmembrane collagens (i.e., collagens XIII, XVII, XXIII and XXV) in mouse brain. Using quantitative PCR and in situ hybridization (ISH), we demonstrate both region- and cell-specific expression of these unique collagens in the developing brain. For the two most highly expressed transmembrane collagens (i.e., collagen XXIII and XXV), we demonstrate that they are expressed by select subsets of neurons in different parts of the brain. For example, collagen XXIII is selectively expressed by excitatory neurons in the mitral/tufted cell layer of the accessory olfactory bulb (AOB) and by cells in the inner nuclear layer (INL) of the retina. On the other hand, collagen XXV, which is more broadly expressed, is generated by subsets of excitatory neurons in the dorsal thalamus and midbrain and by inhibitory neurons in the retina, ventral thalamus and telencephalon. Not only is col25a1 expression present in retina, it appears specifically enriched in retino-recipient nuclei within the brain (including the suprachiasmatic nucleus (SCN), lateral geniculate complex, olivary pretectal nucleus (OPN) and superior colliculus). Taken together, the distinct region- and cell-specific expression patterns of transmembrane collagens suggest that this family of unconventional collagens may play unique, yet-to-be identified roles in brain development and function.