Biology subjects

Blackshaw, S.

Publications and source records attributed to Blackshaw, S..

At least 19 recordsLinked to original sources

bioRxiv2026

An NR2F1-dependent retinoic acid network controls retinal specialization in mice and reveals foveal hypoplasia in patients with BBSOAS

The molecular programs that establish specialized retinal regions during development are essential for high-acuity vision, yet how their disruption contributes to human visual disorders remains poorly understood. Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), caused by pathogenic variants in NR2F1 and characterized by visual impairment, provides an opportunity to investigate these mechanisms. Using single-cell RNA sequencing of three complementary Nr2f1 mouse models, including two carrying patient-specific mutations, we identified a shared Nr2f1-dependent transcriptional program enriched in retinoic acid (RA) pathway genes. Loss or mutation of Nr2f1 disrupted the spatial organization of RA signaling, most prominently by expanding the dorso-equatorial Cyp26a1 expression domain into ventral retina and reducing ventral determinants such as Vax2. These molecular changes were associated with altered dorso-ventral distribution of S- and M-opsin-expressing cone photoreceptors. We further demonstrate that human NR2F1 binds a conserved regulatory region upstream of CYP26A1, supporting its direct role in regulating local RA availability. Finally, high-resolution optical coherence tomography in individuals with BBSOAS revealed reproducible foveal abnormalities, including a smaller and shallower foveal pit and increased central retinal thickness, consistent with foveal hypoplasia. These findings uncover a previously unrecognized retina-intrinsic component of BBSOAS visual pathology and establish an NR2F1-RA/CYP26A1 regulatory axis linking developmental retinal regionalization to human foveal specialization.

developmental biology↗

bioRxiv2026

An Integrated Multi-omics Single Cell Atlas of the Human RPE and Choroid

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.

genomics↗

bioRxiv2026

Isolation and Characterization of Extracellular Vesicles from Mouse Retina Tissue

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.

cell biology↗

bioRxiv2026

The extreme diversity of retinal amacrine cells has deep evolutionary roots

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 co-evolution. 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.

neuroscience↗

bioRxiv2026

Sox8 and Sox9 regulate differentiation and nuclear positioning of retinal Muller glia.

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 Muller 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 Muller glia specification, but caused radial displacement of Muller glia nuclei into the outer retina and modest changes in glial gene expression. Loss of Sox8/9 in mature Muller 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 Muller glia.

neuroscience↗

bioRxiv2025

The long noncoding RNA Peanut (Gm11454) promotes neurogenesis and rod photoreceptor differentiation during postnatal retinal development.

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.

developmental biology↗

bioRxiv2025

Blocking apoptosis promotes survival and alters developmental dynamics of human retinal ganglion cells in retinal organoids

Retinal ganglion cells (RGCs) are the projection neurons that transmit visual information from 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 the timing and cell-type-specificity of 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. To investigate how the apoptotic waves affect retinal development, we differentiated human BAX/BAK double mutant organoids deficient in apoptosis. In these mutants, RGC lifespan and survival increased, while RGC neurogenesis and maturation were delayed. Thus, developmental apoptosis controls not only the quantity of RGCs but also their developmental dynamics. Together, 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 like glaucoma.

developmental biology↗

bioRxiv2025

Comparative single-cell multiomic analysis reveals evolutionarily conserved and species-specific cellular mechanisms mediating natural retinal aging.

Biological age is a major risk factor in the development of common degenerative retinal diseases such as age-related macular degeneration and glaucoma. To systematically characterize molecular mechanisms underlying retinal aging, we performed integrated single- cell RNA- and ATAC-Seq analyses of the retina and retinal pigment epithelium (RPE) across the natural lifespan in zebrafish, mice, and humans. By profiling gene expression and chromatin accessibility, we identified extensive cell type- and species-specific aging-dependent changes, with a much smaller number of broadly expressed and conserved genes that include regulators of inflammation and autophagy. We constructed predictive aging clocks for retinal cell types and observed dynamic, reversible shifts in cellular age following acute injury. Spatial transcriptomic analysis revealed region-specific aging signatures and proximity effects, with Muller glia exhibiting pro-rejuvenating influences on neighboring neurons. Targeted Muller glia-specific induction of Yamanaka factors reduced molecular age in rod photoreceptors and bipolar cells without altering glial age. Our findings define conserved and divergent regulatory and signaling pathways mediating retinal aging, highlighting Muller glia as potential therapeutic targets for combating age-associated retinal dystrophies.

neuroscience↗

bioRxiv2025

Sleep pressure is differentially regulated by molecularly distinct subtypes of Lhx6-positive and Lhx6-negative neurons of the zona incerta.

Sleep pressure, the accumulating drive towards sleep during wakefulness, is shaped by Lhx6-positive GABAergic neurons in the zona incerta (ZI). Here, we show that these neurons are broadly activated both by spontaneous and experimentally-elevated sleep pressure, and remain active for hours into recovery sleep. Activation is stronger in anterior ZI and varies across molecularly defined subgroups: Nkx2-2-positive neurons respond vigorously, whereas Calb2-positive neurons respond weakly. We also identify Lhx6-negative/Slc32a1-positive GABAergic ZI neurons with distinct sleep pressure responses. Selective genetic ablation of Nkx2-2 in Lhx6-positive neurons reduces the number of Lhx6-positive neurons, alters their distribution, blunts sleep pressure-induced activation of both Lhx6-positive and negative cells, and increases sleep duration. These findings show that developmentally specified, molecularly heterogeneous Lhx6-positive ZI neurons form a key hub for regulating sleep homeostasis, and offer new insight into the circuitry that controls sleep pressure.

neuroscience↗

bioRxiv2025

Ptbp1 is not required for retinal neurogenesis and cell fate specification.

The RNA-binding protein Ptbp1 has been proposed as a master regulator of neuronal fate, repressing neurogenesis through its effects on alternative splicing and miRNA maturation. While prior studies using RNA interference suggested that Ptbp1 loss promotes neurogenesis, recent genetic studies have failed to replicate glia-to-neuron conversion following Ptbp1 loss of function. To evaluate the role of Ptbp1 in developmental neurogenesis in vivo, we conditionally disrupted Ptbp1 in retinal progenitors. Ptbp1 was robustly expressed in both retinal progenitors and Muller glia but absent from postmitotic neurons, and efficient loss of function in mutant animals was confirmed using immunostaining for Ptbp1. Furthermore, bulk RNA-Seq at E16 revealed accelerated expression of late-stage progenitor and photoreceptor-specific genes and altered splicing patterns in Ptbp1 mutants, including increased inclusion of rod photoreceptor-specific exons. However, we observed no defects in retinal lamination, progenitor proliferation, or cell fate specification in mature retina. ScRNA-Seq of mature mutant retinas revealed only modest transcriptional changes which partially recapitulate alterations seen following selective deletion of Ptbp1 in mature glia. Our findings demonstrate that Ptbp1 is dispensable for retinal cell fate specification and suggest that its proposed role as a central repressor of neurogenesis should be reevaluated.

neuroscience↗

bioRxiv2025

Overexpression of Meis factors in late-stage retinal progenitors yields complex effects on temporal patterning and neurogenesis.

The vertebrate retina serves as a model for studying neurogenesis and cell fate specification, with retinal progenitor cells following a tightly regulated temporal sequence to generate distinct cell types. Meis1 and Meis2 are transcription factors implicated in early retinal development, but their role in late-stage RPCs remains poorly understood. Here, we investigate whether Meis1 and Meis2 overexpression in postnatal mouse RPCs can alter temporal identity and induce early-born cell types. Using electroporation and single-cell RNA sequencing, we find that while these factors modestly upregulate early-stage gene regulatory network components, they do not repress late-stage transcription factors or induce early-born retinal cells. Meis1 overexpression reduces proliferation and inhibits neurogenesis, whereas Meis2 overexpression accelerates neurogenic progression without altering fate commitment. Our findings suggest that overexpression of Meis1 and Meis2 modulate largely non-overlapping aspects of temporal identity and neurogenic competence but are insufficient to fully reprogram late-stage progenitors. These results have implications for regenerative strategies aimed at reprogramming retinal cells for therapeutic purposes.

neuroscience↗

bioRxiv2025

Heterochronic transcription factor expression drives cone-dominant retina development in 13-lined ground squirrels.

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 (scRNA-Seq) 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.

neuroscience↗

bioRxiv2025

JACUZI-SD: An automated, high-throughput, minimally stressful approach to sleep depriving larval zebrafish.

While sleep deprivation broadly disrupts health and well-being, the neural and molecular mechanisms that signal increased sleep pressure remain poorly understood. A key obstacle to progress is the fact that traditional methods for inducing sleep deprivation (SD) in animal models often introduce confounding stress or disrupt circadian rhythms. Here, we present JACUZI-SD (Jetting Automated Currents Under Zebrafish to Induce Sleep Deprivation), a fully automated, high-throughput system designed to induce sleep deprivation in larval zebrafish with minimal stress. By delivering randomized water pulses via a custom milli-fluidic device integrated with a 96-well plate and controlled by an Arduino, JACUZI-SD promotes wakefulness during the natural dark cycle without the stress associated with existing SD methods. Our results demonstrate that JACUZI-SD reduces total sleep time by 41-64% and elicits a robust rebound sleep characterized by increased sleep bout length following deprivation. Importantly, this method avoids activating the hypothalamic-pituitary-interrenal (HPI) stress axis, as evidenced by reduced stress marker expression compared to other deprivation methods. Additionally, the system reliably activates established sleep pressure pathways, including the upregulation of galanin in the neurosecretory preoptic area, while also revealing biologically relevant inter-individual variability in homeostatic rebound responses. JACUZI-SD provides a powerful, minimally invasive platform for dissecting the neural and molecular underpinnings of sleep homeostasis in vertebrates.

neuroscience↗

bioRxiv2025

DIO3 coordinates photoreceptor development timing and fate stability in human retinal organoids

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 (PR) density, and adoption of L/M cone fate characteristics by S cones and rods. Our multiomics and chimeric organoid experiments show that cell autonomous and non-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 to a probabilistic, cell-intrinsic mechanism. Based on our findings, we propose an hourglass hypothesis, in which the proportion of progenitors to neurons decreases over time to relieve TH degradation, which triggers development of PR 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.

developmental biology↗

bioRxiv2025

Cigarette smoke and biological age induce degenerative heterogeneity in retinalpigment epithelium

Environmental exposure such as cigarette smoke induces epigenetic changes that can induce degenerative heterogeneity and accelerate aging. In early 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 the strongest environmental risk factor for AMD, we hypothesized that cigarette smoke induces degenerative RPE heterogeneity through epigenetic changes that are distinct from aging, and that with aging, the RPE becomes vulnerable to cigarette smoke insult. We administered cigarette smoke condensate (CSC) intravitreally to young and aged mice and performed snRNA-seq and snATAC-seq on the RPE/choroid. This analysis identified separate cell clusters corresponding to healthy and abnormal, dedifferentiated RPE in both aged vehicle-treated and young CSC-treated mice. The dedifferentiated RPE were characterized by a global decrease in chromatin accessibility and decreased expression of genes in functional categories that were linked to hallmarks of aging. Notably, young, dedifferentiated RPE also exhibited a compensatory upregulation of hallmarks of aging-related genes, specifically those related to mitochondrial function and proteostasis. In contrast, aged dedifferentiated RPE did not express these compensatory changes, and did not survive CSC treatment, as experimentally verified with TUNEL labeling. These changes are relevant to early AMD because we identified through scRNA-seq, similar dedifferentiated and healthy macular RPE clusters in a donor who smoked and another with early AMD, but not from a nonsmoker. Degenerative cellular heterogeneity can include an abnormal cluster that jeopardizes cell survival and may represent an additional hallmark of ocular aging.

neuroscience↗

bioRxiv2025

MetaLigand: A database for predicting non-peptide ligand mediated cell-cell communication

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, an R-based and web-accessible tool designed to infer NPL production and predict 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 to improve prediction accuracy. Comparisons with existing tools demonstrate MetaLigands superior ability to account for complex biogenesis pathways and model NPL abundance across diverse tissues and cell types. Furthermore, analysis of single-nucleus 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. Finally, MetaLigand supports single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics data, enabling the visualization of predicted NPL production levels and heterogeneity at single-cell resolution.

bioinformatics↗

bioRxiv2025

Decoding Gene Networks Controlling Hypothalamic and Prethalamic Neuron Development.

Neuronal subtypes derived from the embryonic hypothalamus and prethalamus regulate many essential physiological processes, yet the gene regulatory networks controlling their development remain poorly understood. Using single-cell RNA- and ATAC-sequencing, we analyzed mouse hypothalamic and prethalamic development from embryonic day 11 to postnatal day 8, profiling 660,000 cells in total. This identified key transcriptional and chromatin dynamics driving regionalization, neurogenesis, and differentiation. This identified multiple distinct neural progenitor populations, as well as gene regulatory networks that control their spatial and temporal identities, and their terminal differentiation into major neuronal subtypes. Integrating these results with large-scale genome-wide association study data, we identified a central role for transcription factors controlling supramammillary hypothalamic development in a broad range of metabolic and cognitive traits. Recurring cross-repressive regulatory relationships were observed between transcription factors that induced prethalamic and tuberal hypothalamic identity on the one hand and mammillary and supramammillary hypothalamic identity on the other. In postnatal animals, Dlx1/2 was found to severely disrupt GABAergic neuron specification in both the hypothalamus and prethalamus, resulting in a loss of inhibition of thalamic neurons, hypersensitivity to cold, and behavioral hyperactivity. By identifying core gene regulatory networks controlling the specification and differentiation of major hypothalamic and prethalamic neuronal cell types, this study provides a roadmap for future efforts aimed at preventing and treating a broad range of homeostatic and cognitive disorders.

neuroscience↗

bioRxiv2025

Resolving forebrain developmental organisation by analysis of differential growth patterns

The forebrain is the most complex region of the vertebrate CNS, and its developmental organisation is controversial. We fate-mapped the embryonic chick forebrain using lipophilic dyes and Cre-recombination lineage tracing, and built a 4D model of brain growth. We reveal modular patterns of anisotropic growth, ascribed to progenitor regions through multiplex HCR. Morphogenesis is dominated by directional growth towards the eye, more isometric expansion of the prethalamus and dorsal telencephalon, and anterior movement of ventral cells into the hypothalamus. Fate conversion experiments in chick and comparative gene expression analysis in chick and mouse support placement of the hypothalamus ventral to structures extending from the telencephalon up to and including the zona limitans intrathalamica (ZLI), with the dorsoventral axis becoming distorted at the base of the ZLI. Our findings challenge the widely accepted prosomere model of forebrain organisation, and we propose an alternative tripartite hypothalamus model.

developmental biology↗