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Cone photoreceptors in the eye enable color vision, responding to different wavelengths of light according to what opsin pigments they express. Eldred et al. studied organoids that recapitulate the development of the human retina and found that differentiation of cone cells into their tuned subtypes was regulated by thyroid hormone. Cones expressing short-wavelength (S) opsin developed first, and cones expressing long- and medium-wavelength (L/M) opsin developed later. The switch toward development of L/M cones depended on thyroid hormone signaling through the nuclear thyroid hormone receptor. Science , this issue p. eaau6348 Human retinal organoids offer an opportunity to study the pathways regulating development of color vision. The mechanisms underlying specification of neuronal subtypes within the human nervous system are largely unknown. The blue (S), green (M), and red (L) cones of the retina enable high-acuity daytime and color vision. To determine the mechanism that controls S versus L/M fates, we studied the differentiation of human retinal organoids. Organoids and retinas have similar distributions, expression profiles, and morphologies of cone subtypes. S cones are specified first, followed by L/M cones, and thyroid hormone signaling controls this temporal switch. Dynamic expression of thyroid hormone–degrading and –activating proteins within the retina ensures low signaling early to specify S cones and high signaling late to produce L/M cones. This work establishes organoids as a model for determining mechanisms of human development with promising utility for therapeutics and vision repair.

The retinal degenerative diseases, which together constitute a leading cause of hereditary blindness worldwide, are largely untreatable. Development of reliable methods to culture complex retinal tissues from human pluripotent stem cells (hPSCs) could offer a means to study human retinal development, provide a platform to investigate the mechanisms of retinal degeneration and screen for neuroprotective compounds, and provide the basis for cell-based therapeutic strategies. In this study, we describe an in vitro method by which hPSCs can be differentiated into 3D retinas with at least some important features reminiscent of a mature retina, including exuberant outgrowth of outer segment-like structures and synaptic ribbons, photoreceptor neurotransmitter expression, and membrane conductances and synaptic vesicle release properties consistent with possible photoreceptor synaptic function. The advanced outer segment-like structures reported here support the notion that 3D retina cups could serve as a model for studying mature photoreceptor development and allow for more robust modeling of retinal degenerative disease in vitro .

Age-related macular degeneration (AMD) is a significant cause of vision loss in the elderly. The extent to which epigenetic changes regulate AMD progression is unclear. Here we globally profile chromatin accessibility using ATAC-Seq in the retina and retinal pigmented epithelium (RPE) from AMD and control patients. Global decreases in chromatin accessibility occur in the RPE with early AMD, and in the retina of advanced disease, suggesting that dysfunction in the RPE drives disease onset. Footprints of photoreceptor and RPE-specific transcription factors are enriched in differentially accessible regions (DARs). Genes associated with DARs show altered expression in AMD. Cigarette smoke treatment of RPE cells recapitulates chromatin accessibility changes seen in AMD, providing an epigenetic link between a known risk factor for AMD and AMD pathology. Finally, overexpression of HDAC11 is partially responsible for the observed reduction in chromatin accessibility, suggesting that HDAC11 may be a potential new therapeutic target for AMD. Age-related macular degeneration (AMD) leads to dysfunctional retinal pigment epithelium (RPE) and vision loss. Here, the authors perform ATAC-seq to study chromatin accessibility and find that differentially accessible regions are enriched for photoreceptor and RPE-specific transcription factors in AMD

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs.

Injury and loss of oligodendrocytes can cause demyelinating diseases such as multiple sclerosis. To improve our understanding of human oligodendrocyte development, which could facilitate development of remyelination-based treatment strategies, here we describe time-course single-cell-transcriptomic analysis of developing human stem cell-derived oligodendrocyte-lineage-cells (hOLLCs). The study includes hOLLCs derived from both genome engineered embryonic stem cell (ESC) reporter cells containing an Identification-and-Purification tag driven by the endogenous PDGFRα promoter and from unmodified induced pluripotent (iPS) cells. Our analysis uncovers substantial transcriptional heterogeneity of PDGFRα-lineage hOLLCs. We discover sub-populations of human oligodendrocyte progenitor cells (hOPCs) including a potential cytokine-responsive hOPC subset, and identify candidate regulatory genes/networks that define the identity of these sub-populations. Pseudotime trajectory analysis defines developmental pathways of oligodendrocytes vs astrocytes from PDGFRα-expressing hOPCs and predicts differentially expressed genes between the two lineages. In addition, pathway enrichment analysis followed by pharmacological intervention of these pathways confirm that mTOR and cholesterol biosynthesis signaling pathways are involved in maturation of oligodendrocytes from hOPCs. Brain myelinating oligodendrocytes are rare and difficult to isolate, which has limited data on their development. Here the authors develop a reporter for scalable purification of human pluripotent stem cell derived oligodendrocyte lineage cells, and use this to map differentiation using single cell RNA-sequencing,

Background DNA methylation plays an important role in regulating gene expression during many biological processes. However, the mechanism of DNA-methylation-dependent gene regulation is not fully understood. Here, we explore two possible DNA methylation regulatory mechanisms with opposite modes of gene expression regulation. Results By comparing the genome-wide methylation and expression patterns in different tissues, we find that majority of tissue-specific differentially methylated regions (T-DMRs) are negatively correlated with expression of their associated genes (negative T-DMRs), consistent with the classical dogma that DNA methylation suppresses gene expression; however, a significant portion of T-DMRs are positively correlated with gene expression (positive T-DMRs). We observe that the positive T-DMRs have similar genomic location as negative T-DMRs, except that the positive T-DMRs are more enriched in the promoter regions. Both positive and negative T-DMRs are enriched in DNase I hypersensitivity sites (DHSs), suggesting that both are likely to be functional. The CpG sites of both positive and negative T-DMRs are also more evolutionarily conserved than the genomic background. Interestingly, the putative target genes of the positive T-DMR are enriched for negative regulators such as transcriptional repressors, suggesting a novel mode of indirect DNA methylation inhibition of expression through transcriptional repressors. Likewise, two distinct sets of DNA sequence motifs exist for positive and negative T-DMRs, suggesting that two distinct sets of transcription factors (TFs) are involved in positive and negative regulation mediated by DNA methylation. Conclusions We find both negative and positive association between T-DMRs and gene expression, which implies the existence of two different mechanisms of DNA methylation-dependent gene regulation.

Complement component C3 mediates pathology in CNS neurodegenerative diseases. Here we use scRNAseq of sorted C3-reporter positive cells from mouse brain and optic nerve to characterize C3 producing glia in experimental autoimmune encephalomyelitis (EAE), a model in which peripheral immune cells infiltrate the CNS, causing reactive gliosis and neuro-axonal pathology. We find that C3 expression in the early inflammatory stage of EAE defines disease-associated glial subtypes characterized by increased expression of genes associated with mTOR activation and cell metabolism. This pro-inflammatory subtype is abrogated with genetic C3 depletion, a finding confirmed with proteomic analyses. In addition, early optic nerve axonal injury and retinal ganglion cell oxidative stress, but not loss of post-synaptic density protein 95, are ameliorated by selective deletion of C3 in myeloid cells. These data suggest that in addition to C3b opsonization of post synaptic proteins leading to neuronal demise, C3 activation is a contributor to reactive glia in the optic nerve. Excessive complement C3 causes synaptic stripping and neurodegeneration. Here, the authors used the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and single-cell RNA sequencing to show that C3 expression defines disease-associated reactive glia. C3 deletion abrogated these pro-inflammatory glia and protected neurons.

The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions.

Aquaporin 4 is absent from astrocytes in the rodent optic nerve head, despite high expression in the retina and myelinated optic nerve. The purpose of this study was to quantify regional aquaporin channel expression in astrocytes of the porcine and human mouse optic nerve (ON). Ocular tissue sections were immunolabeled for aquaporins 1(AQP1), 4(AQP4), and 9(AQP9), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and alpha-dystroglycan (αDG) for their presence in retina, lamina, myelin transition zone (MTZ, region just posterior to lamina) and myelinated ON (MON). Semi- quantification of AQP4 labeling & real-time quantitative PCR (qPCR) data were analyzed in retina and ON tissue. Porcine and control human eyes had abundant AQP4 in Müller cells, retinal astrocytes, and myelinated ON (MON), but minimal expression in the lamina cribrosa. AQP1 and AQP9 were present in retina, but not in the lamina. Immunolabeling of GFAP and αDG was similar in lamina, myelin transition zone (MTZ) and MON regions. Semi-quantitative AQP4 labeling was at background level in lamina, increasing in the MTZ, and highest in the MON (lamina vs MTZ, MON; p≤0.05, p≤0.01, respectively). Expression of AQP4 mRNA was minimal in lamina and substantial in MTZ and MON, while GFAP mRNA expression was uniform among the lamina, MTZ, and MON regions. Western blot assay showed AQP4 protein expression in the MON samples, but none was detected in the lamina tissue. The minimal presence of AQP4 in the lamina is a specific regional phenotype of astrocytes in the mammalian optic nerve head.

Previous studies of neuronal survival have primarily focused on identifying intrinsic mechanisms controlling the process. This study explored how intercellular communication contributes to retinal ganglion cell (RGC) survival following optic nerve crush based on single-cell RNA-seq analysis. We observed transcriptomic changes in retinal cells in response to the injury, with astrocytes and Müller glia having the most interactions with RGCs. By comparing RGC subclasses characterized by distinct resilience to cell death, we found that the high-survival RGCs tend to have more ligand-receptor interactions with neighboring cells. We identified 47 interactions stronger in high-survival RGCs, likely mediating neuroprotective effects. We validated one identified target, the μ-opioid receptor (Oprm1), to be neuroprotective in three retinal injury models. Although the endogenous Oprm1 is preferentially expressed in intrinsically photosensitive RGCs, its neuroprotective effect can be transferred to other subclasses by pan-RGC overexpression of Oprm1. Lastly, manipulating the Oprm1 activity improved visual functions in mice. How the neighboring cells contribute to the survival and functions of neuronal cells remains elusive. Here, authors identified the cell-cell interactions between retinal ganglion cells (RGCs) and other cells after RGC injury and discovered the μ-opioid receptor promotes RGC resilience.