This protocol provides a standardized workflow for the isolation of extracellular vesicles (EVs) from mouse retinal tissue, and includes an assessment of EV size and concentration, marker expression, and EV visualization in accordance with the International Society for Extracellular Vesicles Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. Most retinal EV studies rely on cell culture, which may not fully capture in vivo biology. Our approach more accurately reflects physiological and pathological EV states in vivo by enabling the extraction of EVs from intact retinal tissue. This method addresses a key gap in the field by providing a reproducible and rigorous protocol for studying retinal EVs in a biologically relevant context.

Sleep pressure is regulated not only by circadian rhythms, but also by sleep homeostasis, an activity-dependent process that dissipates during sleep. Recent work implicates Lhx6-positive GABAergic neurons of the zona incerta (ZI) in regulating sleep pressure, but their precise role remains unclear. Using sleep deprivation and HiPlex single-molecule fISH, we show that Lhx6-positive ZI neurons are broadly activated by both natural and induced increases in sleep pressure and remain active for more than 3 h into recovery sleep. Anterior Lhx6-positive neurons showed stronger activation. Fos induction differed across molecularly distinct subpopulations, with Nkx2-2-positive cells showing robust responses and Calb2-positive cells showing reduced activation. We also identified distinct sleep pressure-responsive Lhx6-negative Slc32a1-positive GABAergic ZI subpopulations. Finally, intersectional genetic loss of Nkx2-2 reduced and redistributed Lhx6-positive neurons, blunted their activation, and increased total sleep time. These findings reveal a central, heterogeneous role for Lhx6-positive ZI neurons in sleep homeostasis.

Inadequate sleep is common and broadly disrupts well-being. A fundamental challenge to modeling sleep deprivation (SD) experimentally is the introduction of confounds such as stress or circadian manipulation. We therefore developed JACUZI-SD (Jetting Automated Currents Under Zebrafish to Induce Sleep Deprivation), an automated, high-throughput, and minimally stressful system to SD larval zebrafish during the dark cycle. JACUZI-SD delivers randomized water pulses via a custom milli-fluidic device integrated with a 96-well plate, reducing total sleep time by 41-64% and eliciting a rebound sleep characterized by an 11% increase in total sleep time and 29% increase in sleep bout length. Water pulses are minimally stressful, eliciting less fosab expression in the hypothalamic-pituitary-interrenal (HPI) stress axis compared to other SD methods. Additionally, we observe galanin induction, an established response to heightened sleep pressure. JACUZI-SD provides a powerful, minimally invasive platform for dissecting the neural underpinnings and biological variability of sleep homeostasis in vertebrates.

Temporal patterning of retinal progenitor cells governs the sequential generation of retinal cell types, with gliogenesis occurring late in development. Sox8 and Sox9, members of the SoxE transcription factor family, are highly expressed in late-stage retinal progenitor cells and mature Müller glia, yet their functional roles remain incompletely defined. Here we employed gain- and loss-of-function approaches, single-cell multiomic profiling, and injury models to investigate Sox8/9 function. Overexpression of SOX8 and/or SOX9 in early-stage retinal progenitor cells suppressed early-born cell fates and promoted photoreceptor generation, consistent with a role in late-stage temporal identity. Conversely, conditional deletion of Sox8 and/or Sox9 in late-stage progenitors did not impair Müller glia specification, but caused radial displacement of Müller glia nuclei into the outer retina and modest changes in glial gene expression. Loss of Sox8/9 in mature Müller glia modestly increased proliferation post-injury without inducing neurogenic competence. These findings suggest that Sox8/9 are dispensable for gliogenesis and repression of neurogenic competence, but are essential for proper laminar positioning and maturation of retinal Müller glia.

For over three decades, the prosomere model has influenced our understanding of vertebrate brain development by proposing that the forebrain consists of several transverse segments, termed prosomeres. However, advances in molecular genetics and imaging have challenged key assumptions of this model, including the existence of clear segmental boundaries and the positioning of forebrain axes. Here we critically review evidence from gene expression, fate-mapping, and morphogenesis studies, finding limited support for prosomeric segmentation and questioning the grouping of the hypothalamus with the telencephalon. We propose the tripartite hypothalamus model as an alternative framework that better aligns with recent data, restoring the hypothalamus's relationship to the diencephalon and recognizing the anterior forebrain as a uniquely complex, non-segmented unit with a posterior boundary within the diencephalon. These findings suggest a need to revise our understanding of forebrain structure, patterning, and evolution.

In the central region of the human retina, the high-acuity foveola is notable for its dense packing of green (M) and red (L) cones and absence of blue (S) cones. To identify mechanisms that pattern cones in the foveola, we examined human fetal retinas and differentiated retinal organoids. During development, sparse S-opsin-expressing cones are initially observed in the foveola. Later in fetal development, the foveola contains a mix of cones that either coexpress S-and M/L-opsins or exclusively express M/L-opsin. In adults, only M/L cones are present. Two signaling pathway regulators are highly and continuously expressed in the central retina: Cytochrome P450 26 subfamilyA member 1 (CYP26A1), which degrades retinoic acid (RA) and Deiodinase 2 (DIO2), which promotes thyroid hormone (TH) signaling. Both CYP26A1 mutant organoids and high RA conditions increased the number of S cones and reduced the number of M/L cones in retinal organoids. In contrast, sustained TH signaling promoted the generation of M/L-opsin-expressing cones and induced M/L-opsin expression in S-opsin-expressing cones, showing that cone fate is plastic. Our data suggest that CYP26A1 degrades RA to specify M/L cones and limit S cones and that continuous DIO2 expression sustains high levels of TH to transition S-opsin-expressing cones into M/L cone fate, resulting in the foveola containing only M/L cones. Given the vulnerability of the foveola in macular degeneration and other retinal disorders, these findings provide a mechanistic framework for engineering organoids for therapeutic applications.

The mechanisms governing the generation of neuronal subtypes at distinct times and proportions during human retinal development are poorly understood. While thyroid hormone (TH) signaling specifies cone photoreceptor subtypes, how this regulation changes over time remains unclear. To address this question, we studied the expression and function of type 3 iodothyronine deiodinase (DIO3), an enzyme that degrades TH, in human retinal organoids. We show that DIO3 is a master regulator of human photoreceptor developmental timing and cell fate stability. DIO3 is highly expressed in retinal progenitor cells (RPCs) and decreases as these cells asynchronously differentiate into neurons, progressively reducing TH degradation and increasing TH signaling. DIO3 mutant organoids display precocious development of S cones, L/M cones, and rods; increased photoreceptor density; and subpopulations of photoreceptors that coexpress different opsin proteins. Our multiomics and chimeric organoid experiments show that cell-autonomous and non-cell-autonomous mechanisms locally coordinate and maintain DIO3 expression and TH signaling levels among cells. Computational modeling reveals a mechanism that couples TH levels and fate specification, providing robustness to photoreceptor development as compared with a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass-like mechanism in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, triggering development of photoreceptor subtypes at specific times. Our study identifies how local regulation of thyroid hormone signaling influences neural cell fate specification, which may be a consideration for designing regenerative therapies.

Retinal ganglion cells (RGCs) are the projection neurons connecting the retina to the brain. In many species, a substantial proportion of RGCs are eliminated by programmed cell death during development to regulate their final number, but how cell death impacts human RGC development remains poorly understood. Here, we characterized cell death in human fetal retinas and retinal organoids. Both retinas and organoids exhibited two waves of apoptosis: an early wave targeting neurogenic retinal progenitor cells and neuronal precursors and a late wave affecting RGCs and other neurons. Additionally, organoids displayed a distinct wave of necrosis. Blocking apoptosis in organoids via BAX/BAK double knockout improved RGC survival but delayed RGC neurogenesis and maturation. Our results highlight the roles of apoptosis in human RGC development and the challenges in retinal organoid design. Addressing these limitations will improve the utility of organoids for studying human retinal development and modeling optic neuropathies such as glaucoma.

Cigarette smoking induces epigenetic changes that can cause degenerative heterogeneity with aging and disease. In disease such as age--related macular degeneration (AMD), the leading worldwide cause of blindness among the elderly, retinal pigment epithelial (RPE) cell heterogeneity is a key change. Since smoking is a powerful risk factor for AMD, we hypothesized that smoke induces epigenetic--mediated degenerative RPE heterogeneity. We administered cigarette smoke condensate (CSC) to young and aged mice. Using snRNA-seq and single nuclear ATAC sequencing, we identified distinct healthy and dedifferentiated RPE clusters in both aged vehicle-and young CSC--treated mice. Dedifferentiated RPE had globally decreased chromatin accessibility and expression of genes linked to "hallmarks of aging." Notably, young, dedifferentiated RPE also exhibited a compensatory upregulation of hallmarks of aging--related genes including mitochondrial function and proteostasis while aged dedifferentiated RPE did not, which decreased their survival following CSC treatment, as experimentally verified with TUNEL labeling. Similar populations of dedifferentiated and healthy RPE were identified both in mice exposed to cigarette smoke for 4 mo and in macular RPE from a donor who smoked and another with early AMD, but not from a nonsmoker donor. Degenerative cellular heterogeneity that includes an abnormal cluster can jeopardize cell survival and represents a hallmark of ocular aging.

Evolutionary adaptation to diurnal vision in ground squirrels has led to the development of a cone-dominant retina, in stark contrast to the rod-dominant retinas of most mammals. The molecular mechanisms driving this shift remain largely unexplored. Here, we perform single-cell RNA sequencing and chromatin accessibility profiling (scATAC-Seq) across developmental retinal neurogenesis in the 13-lined ground squirrel (13LGS) to uncover the regulatory basis of this adaptation. We find that 13LGS cone photoreceptors arise not only from early-stage neurogenic progenitors, as seen in rod-dominant species like mice, but also from late-stage neurogenic progenitors. This extended period of cone generation is driven by a heterochronic shift in transcription factor expression, with cone-promoting factors such as Onecut2, Pou2f1, and Zic3 remaining active in late-stage progenitors, and factors that promote cone differentiation such as Thrb, Rxrg, and Mef2c expressed precociously in late-stage neurogenic progenitors. Functional analyses reveal that Zic3 and Mef2c are sufficient to promote cone and repress rod photoreceptor-specific gene expression and act through species-specific regulatory elements that drive their expression in late-stage progenitors. These results demonstrate that modifications to gene regulatory networks underlie the development of cone-dominant retinas and provide insight into mechanisms of sensory adaptation and potential strategies for cone photoreceptor regeneration in vision disorders.

Amacrine cells (ACs) comprise a heterogeneous class of inhibitory neurons in the vertebrate retina, exhibiting morphological and functional complexity rivaling that of cortical interneurons. Here, we integrate single-cell and single-nucleus transcriptomic atlases from 24 vertebrate species to reconstruct the evolutionary origins of this extreme diversity. We identify 42 orthologous AC types (oACs), most of which exhibit a one-to-one correspondence across amniotes and, in many cases, across vertebrates. While core molecular identities are conserved, AC types vary in abundance and gene expression across species, likely reflecting adaptations to distinct visual ecologies. AC diversity scales with that of retinal ganglion cells (RGCs), indicative of coevolution. Finally, we suggest that ACs arose from an AC-RGC hybrid precursor, with glycinergic ACs diverging early in vertebrate evolution, followed by a bifurcation between RGCs and GABAergic ACs. Together, these findings establish a unified evolutionary framework for understanding the diversity, development, and function of a class of inhibitory neurons across vertebrates.

The retinal pigment epithelium and choroid are critical for supporting the function and maintaining the homeostasis of the outer retina, and their dysfunction underlies a range of inherited and complex ocular diseases. To comprehensively characterize the cellular, transcriptomic, and epigenomic heterogeneity and dynamics within these tissues, we assembled an integrated multi-omics reference atlas comprising 719,813 single-cell/single-nucleus transcriptomes and 234,007 snATAC-seq profiles from 102 ancestrally diverse donors spanning 0 to 99 years of age, including cells from both the macula and periphery. This atlas resolves 48 distinct cell types or states and catalogs 448,567 open chromatin regions. Specifically, we resolved five distinct RPE subpopulations organized along a central-to-peripheral spatial axis, alongside two distinct stress/senescence states. We reconstructed the transcriptomic and epigenetic zonation of endothelial cells and expanded choroidal stromal heterogeneity by characterizing 11 fibroblast and two pericyte types. Age-associated compositional analysis revealed a significant fractional depletion of melanocytes, PI16+ fibroblasts, and venule endothelial cells with age, alongside a modest relative loss of central RPE and a corresponding increase in far-peripheral RPE. Cell-type-specific aging transcriptomics uncovered shared pathways related to inflammatory responses alongside distinct cell-type-specific signatures. Notably, significant age-associated epigenetic changes concentrated in the macula during the transition from early-to-middle adulthood and remained stable into old age, with transcription factors from the AP-1/bZIP family emerging as the dominant enriched motifs. Finally, integrating this atlas with AMD GWAS data provides novel variant-to-gene evidence implicating LIPG and COL4A3 in AMD pathogenesis. Together, this multi-omics atlas serves as both an invaluable community reference and a powerful discovery engine that translates genetic risk signals into localized target cells and candidate mechanisms, laying a foundation for understanding RPE/choroid biology in health and disease.

Retinal ganglion cell (RGC) axons form the optic nerve (ON). Numerous age-related ON diseases, including glaucoma, the second most common cause of worldwide blindness, result from multiple RGC stressors. Nearly all ON astrocytes in the optic nerve head (ONH): the junctional region between the ON and the retina in young-adult rodents expresses the homeodomain only (Hopx) protein. Hopx(+) ONH astrocytes are depleted during aging. ONH primary cultures which include Hopx(+) astrocytes secrete extracellular vesicles (ONH-EVs) which selectively enhance RGC survival and neurite extension in culture, while extracellular vesicles (EVs) secreted from distal ON cultures lacking Hopx(+) astrocytes do not. ONH-EVs also enhance RGC survival in vivo in a rodent model of glaucoma. Combining rat ONH single-cell (scRNA-seq) sequencing with EV proteomic analysis, we identified ONH-Hopx(+) astrocyte secreted factors. We interrogated the online Broad institute scRNA-seq database for rat RGC gene expression in control animals and following rodent ON crush, an RGC stress model, to correlate ONH-astrocyte secreted factors with RGC gene expression changes. Following stress, RGCs upregulate the complementary pathways involving Hopx(+) astrocytic-associated factors, suggesting reciprocal communication. Using a highly selective transgenic Hopx-cre ONH knockdown strategy, we demonstrate that eliminating Hopx(+) astrocytes also results in upregulation of RGC stress responses. Our results implicate age-related loss of young ONH-astrocytes as a crucial factor in the development of age-related optic nerve diseases, and discuss replacing ONH associated factors as a paradigm shift for ON disease treatment.

Synovial joints are complex organs composed of specialized tissues whose coordinated formation is essential for proper joint function. The cellular composition and regulatory programs governing late synovial joint development are less well characterized as compared to earlier stages of joint development. Here, we generated single-cell transcriptomic data of Growth differentiation factor 5 (Gdf5)-lineage cells from the embryonic mouse knee joint at embryonic day 17.5 and integrated these data with those from earlier developmental stages to infer transcriptional state progression across joint development. We identified nine distinct Gdf5-lineage populations corresponding to major joint tissues, including articular chondrocytes, superficial lining cells, ligament-tenogenic progenitors, synovial fibroblasts, and progenitor populations, and we validated their localization within the embryonic joint. We found a meniscus-associated progenitor state characterized by low expression of canonical chondrocyte markers and high expression of pleiotrophin (Ptn), distinguishing it from conventional articular chondrocytes. In addition, we found that Col22a1 and tetraspanin 15 (Tspan15) mark a transcriptionally and spatially distinct superficial lining cell population. To identify potential regulatory mechanisms underlying articular chondrocyte development, we reconstructed dynamic gene regulatory networks along the progenitor-to-chondrocyte trajectory, revealing a transition from a common regulatory state to cell type-specific regulatory program at later stages. In summary, our study provides data that improves our understanding of the developmental transcriptional programs that contribute to the major tissues of the synovial joint.

Regulation of neural progenitor temporal identity is critical to control the chronological order of cell birth and generation of cell diversity in the developing central nervous system (CNS). Single-cell RNA sequencing studies have identified transcriptionally distinct early and late temporal identity states in mammalian neural progenitors in multiple CNS regions. This review discusses recent advances in understanding the mechanisms underlying regulation of temporal identity in mammalian neural progenitors, the implications of these findings for glia-to-neuron reprogramming strategies, and their potential therapeutic applications. We highlight potential future directions of research, including integrating temporal identity specification with proneural factor overexpression to enhance reprogramming efficiency and broaden the repertoire of neuronal subtypes generated from reprogrammed mammalian glia.

Retinal Müller glia in cold-blooded vertebrates can reprogram into neurogenic progenitors to replace neurons lost to injury, but mammals lack this ability. While recent studies have shown that transgenic overexpression of neurogenic bHLH factors and glial-specific disruption of NFI family transcription factors and Notch signaling induce neurogenic competence in mammalian Müller glia, induction of neurogenesis in wildtype glia has thus far proven elusive. Here, we report that viral-mediated overexpression of the pluripotency factor Pou5f1 (Oct4) induces transdifferentiation of mouse Müller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single-cell multiomic analysis shows that Pou5f1 overexpression leads to widespread changes in gene expression and chromatin accessibility, inducing activity of both the neurogenic transcription factor Rfx4 and the Yamanaka factors Sox2 and Klf4. This study demonstrates that viral-mediated overexpression of Pou5f1 induces neurogenic competence in adult mouse Müller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

Non-peptide ligands (NPLs), including lipids, amino acids, carbohydrates, and non-peptide neurotransmitters and hormones, play a critical role in ligand-receptor-mediated cell-cell communication, driving diverse physiological and pathological processes. To facilitate the study of NPL-dependent intercellular interactions, we introduce MetaLigand, a tool designed to infer NPL availability and NPL-receptor interactions using transcriptomic data. MetaLigand compiles data for 233 NPLs, including their biosynthetic enzymes, transporter genes, and receptor genes, through a combination of automated pipelines and manual curation from comprehensive databases. The tool integrates both de novo and salvage synthesis pathways, incorporating multiple biosynthetic steps and transport mechanisms. Comparisons with existing tools demonstrate MetaLigand's ability to account for complex biogenesis pathways and model NPL availability across diverse tissues and cell types. Furthermore, analysis of single-nucleus RNA sequencing (RNA-seq) datasets from age-related macular degeneration samples revealed that distinct retinal cell types exhibit unique NPL profiles and participate in specific NPL-mediated pathological cell-cell interactions.

AbstractRetinal Müller glia in cold-blooded vertebrates can reprogram into neurogenic progenitors to replace neurons lost to injury, but mammals lack this ability. While recent studies have shown that transgenic overexpression of neurogenic bHLH factors and glial-specific disruption of NFI family transcription factors and Notch signaling induce neurogenic competence in mammalian Müller glia, induction of neurogenesis in wildtype glia has thus far proven elusive. Here we report that viral-mediated overexpression of the pluripotency factorOct4(Pou5f1) induces transdifferentiation of wildtype mouse Müller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single cell multiomic analysis shows thatOct4overexpression leads to widespread changes in gene expression and chromatin accessibility, inducing activity of both the neurogenic transcription factor Rfx4 and the Yamanaka factors Sox2 and Klf4. This study demonstrates that viral-mediated overexpression of Oct4 induces neurogenic competence in wildtype retinal Muller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

Long noncoding RNAs (lncRNAs) display pervasive expression and function in the developing nervous system. Temporal profiling of gene expression in the retina has demonstrated differential expression of lncRNAs throughout development; however, determinations of lncRNA function during retinal development remain limited. In this study, we identify numerous lncRNAs with dynamic temporal expression and characterize the function of the lncRNA Gm11454, which we have named Peanut . Using overexpression of Peanut in mice retinas, we determine that Peanut promotes rod photoreceptor fate and neurogenesis of retinal progenitor cells (RPCs) via inhibition of Notch signaling and by regulating expression of neighboring gene Tox2 . A novel Peanut knockout mouse model demonstrates that Peanut is required for proper visual function and photoreceptor gene expression. Finally, we determined that Peanut is necessary for proper cell cycle progression and neurogenesis. Our results characterize the function of a novel lncRNA as a regulator of RPC neurogenesis and differentiation and support the importance of lncRNAs in the developing retina.