A single-cell time-lapse of mouse prenatal development from gastrula to birth.

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans1,2. Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb.

QR-1011 restores defective ABCA4 splicing caused by multiple severe ABCA4 variants underlying Stargardt disease.

Stargardt disease type 1 (STGD1), the most common form of hereditary macular dystrophy, can be caused by biallelic combinations of over 2200 variants in the ABCA4 gene. This leads to reduced or absent ABCA4 protein activity, resulting in toxic metabolite accumulation in the retina and damage of the retinal pigment epithelium and photoreceptors. Approximately 21% of all ABCA4 variants that contribute to disease influence ABCA4 pre-mRNA splicing. This emphasizes the need for therapies to restore disrupted ABCA4 splicing and halt STGD1 progression. Previously, QR-1011, an antisense oligonucleotide (AON), successfully corrected splicing abnormalities and restored normal ABCA4 protein translation in human retinal organoids carrying the prevalent disease-causing variant c.5461-10T>C in ABCA4. Here, we investigated whether QR-1011 could also correct splicing in four less common non-canonical splice site (NCSS) variants flanking ABCA4 exon 39: c.5461-8T>G, c.5461-6T>C, c.5584+5G>A and c.5584+6T>C. We administered QR-1011 and three other AONs to midigene-transfected cells and demonstrate that QR-1011 had the most pronounced effect on splicing compared to the others. Moreover, QR-1011 significantly increased full-length ABCA4 transcript levels for c.5461-8T>G and c.5584+6T>C. Splicing restoration could not be achieved in the other two variants, suggesting their more severe effect on splicing. Overall, QR-1011, initially developed for a single ABCA4 variant, exhibited potent splice correction capabilities for two additional severe NCSS variants nearby. This suggests the possibility of a broader therapeutic impact of QR-1011 extending beyond its original target and highlights the potential for treating a larger population of STGD1 patients affected by multiple severe ABCA4 variants with a single AON.

Medioapical contractile pulses coordinated between cells regulate Drosophila eye morphogenesis.

Lattice cells (LCs) in the developing Drosophila retina change shape before attaining final form. Previously, we showed that repeated contraction and expansion of apical cell contacts affect these dynamics. Here, we describe another factor, the assembly of a Rho1-dependent medioapical actomyosin ring formed by nodes linked by filaments that contract the apical cell area. Cell area contraction alternates with relaxation, generating pulsatile changes in cell area that exert force on neighboring LCs. Moreover, Rho1 signaling is sensitive to mechanical changes, becoming active when tension decreases and cells expand, while the negative regulator RhoGAP71E accumulates when tension increases and cells contract. This results in cycles of cell area contraction and relaxation that are reciprocally synchronized between adjacent LCs. Thus, mechanically sensitive Rho1 signaling controls pulsatile medioapical actomyosin contraction and coordinates cell behavior across the epithelium. Disrupting the kinetics of pulsing can lead to developmental errors, suggesting this process controls cell shape and tissue integrity during epithelial morphogenesis of the retina.

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.

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina.

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.

An ON-type direction-selective ganglion cell in primate retina.

To maintain a stable and clear image of the world, our eyes reflexively follow the direction in which a visual scene is moving. Such gaze-stabilization mechanisms reduce image blur as we move in the environment. In non-primate mammals, this behaviour is initiated by retinal output neurons called ON-type direction-selective ganglion cells (ON-DSGCs), which detect the direction of image motion and transmit signals to brainstem nuclei that drive compensatory eye movements1. However, ON-DSGCs have not yet been identified in the retina of primates, raising the possibility that this reflex is mediated by cortical visual areas. Here we mined single-cell RNA transcriptomic data from primate retina to identify a candidate ON-DSGC. We then combined two-photon calcium imaging, molecular identification and morphological analysis to reveal a population of ON-DSGCs in the macaque retina. The morphology, molecular signature and GABA (γ-aminobutyric acid)-dependent mechanisms that underlie direction selectivity in primate ON-DSGCs are highly conserved with those in other mammals. We further identify a candidate ON-DSGC in human retina. The presence of ON-DSGCs in primates highlights the need to examine the contribution of subcortical retinal mechanisms to normal and aberrant gaze stabilization in the developing and mature visual system.

Cell Division Control Protein 42 Facilitates Diabetic Retinopathy Progression by Activating the MEK/ERK Pathway.

Cell division control protein 42 (CDC42) modulates insulin secretion and angiogenesis to participate in the pathology of diabetic complications and retinal vascular-associated diseases. This study intended to explore the role of CDC42 in the progression of diabetic retinopathy, and the underlying mechanism. Human retinal microvascular endothelial cells (hRMECs) were cultured in 5.5 mM glucose (normal glucose) or 25 mM glucose (high glucose; HG) medium, respectively. CDC42 overexpression plasmid and small interference RNA (oe-CDC42 and si-CDC42) or corresponding negative controls (oe-NC and si-NC) were transfected into hRMECs under HG. Then, platelet-activating factor C-16 (C16-PAF) (MEK/ERK pathway activator) was added to si-CDC42 or si-NC transfected hRMECs under HG. Our study showed that HG increased CDC42 mRNA and protein, cell viability, invasive cell count, branch points, and tube length but reduced cell apoptosis in hRMECs. CDC42 upregulation enhanced cell viability, invasive cell count, branch points, tube length, p-MEK, and p-ERK, but attenuated cell apoptosis. Downregulation of CDC42 exhibited opposite trends. In addition, C16-PAF also increased cell viability, invasive cell count, branch points, and tube length, p-MEK, and p-ERK, but retarded cell apoptosis. Notably, C16-PAF diminished the effect of CDC42 downregulation on the above-mentioned functions in hRMECs under HG. Conclusively, CDC42 promotes HG-induced hRMEC viability and invasion, as well as angiogenesis, but inhibits apoptosis by activating the MEK/ERK pathway, which may be responsible for the progression of diabetic retinopathy.

Inhibition of CysLTR1 reduces the levels of aggregated proteins in retinal pigment epithelial cells.

The endosomal-lysosomal system (ELS), which carries out cellular processes such as cellular waste degradation via autophagy, is essential for cell homeostasis. ELS inefficiency leads to augmented levels of damaged organelles and intracellular deposits. Consequently, the modulation of autophagic flux has been recognized as target to remove damaging cell waste. Recently, we showed that cysteinyl leukotriene receptor 1 (CysLTR1) antagonist application increases the autophagic flux in the retinal pigment epithelial cell line ARPE-19. Consequently, we investigated the effect of CysLTR1 inhibition-driven autophagy induction on aggregated proteins in ARPE-19 cells using flow cytometry analysis. A subset of ARPE-19 cells expressed CysLTR1 on the surface (SE+); these cells showed increased levels of autophagosomes, late endosomes/lysosomes, aggregated proteins, and autophagy as well as decreased reactive oxygen species (ROS) formation. Furthermore, CysLTR1 inhibition for 24 h using the antagonist zafirlukast decreased the quantities of autophagosomes, late endosomes/lysosomes, aggregated proteins and ROS in CysLTR1 SE- and SE+ cells. We concluded that high levels of plasma membrane-localized CysLTR1 indicate an increased amount of aggregated protein, which raises the rate of autophagic flux. Furthermore, CysLTR1 antagonist application potentially mimics the physiological conditions observed in CysLTR1 SE+ cells and can be considered as strategy to dampen cellular aging.

Neuronal migration prevents spatial competition in retinal morphogenesis.

The concomitant occurrence of tissue growth and organization is a hallmark of organismal development1-3. This often means that proliferating and differentiating cells are found at the same time in a continuously changing tissue environment. How cells adapt to architectural changes to prevent spatial interference remains unclear. Here, to understand how cell movements that are key for growth and organization are orchestrated, we study the emergence of photoreceptor neurons that occur during the peak of retinal growth, using zebrafish, human tissue and human organoids. Quantitative imaging reveals that successful retinal morphogenesis depends on the active bidirectional translocation of photoreceptors, leading to a transient transfer of the entire cell population away from the apical proliferative zone. This pattern of migration is driven by cytoskeletal machineries that differ depending on the direction: microtubules are exclusively required for basal translocation, whereas actomyosin is involved in apical movement. Blocking the basal translocation of photoreceptors induces apical congestion, which hampers the apical divisions of progenitor cells and leads to secondary defects in lamination. Thus, photoreceptor migration is crucial to prevent competition for space, and to allow concurrent tissue growth and lamination. This shows that neuronal migration, in addition to its canonical role in cell positioning4, can be involved in coordinating morphogenesis.

Perception of color in primates: A conceptual color neurons hypothesis.

Perception of color by humans and other primates is a complex problem, studied by neurophysiology, psychophysiology, psycholinguistics, and even philosophy. Being mostly trichromats, simian primates have three types of opsin proteins, expressed in cone neurons in the eye, which allow for the sensing of color as the physical wavelength of light. Further, in neural networks of the retina, the coding principle changes from three types of sensor proteins to two opponent channels: activity of one type of neuron encode the evolutionarily ancient blue-yellow axis of color stimuli, and another more recent evolutionary channel, encoding the axis of red-green color stimuli. Both color channels are distinctive in neural organization at all levels from the eye to the neocortex, where it is thought that the perception of color (as philosophical qualia) emerges from the activity of some neuron ensembles. Here, using data from neurophysiology as a starting point, we propose a hypothesis on how the perception of color can be encoded in the activity of certain neurons in the neocortex. These conceptual neurons, herein referred to as 'color neurons', code only the hue of the color of visual stimulus, similar to place cells and number neurons, already described in primate brains. A case study with preliminary, but direct, evidence for existing conceptual color neurons in the human brain was published in 2008. We predict that the upcoming studies in non-human primates will be more extensive and provide a more detailed description of conceptual color neurons.

Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation.

How the diverse neural cell types emerge from multipotent neural progenitor cells during central nervous system development remains poorly understood. Recent scRNA-seq studies have delineated the developmental trajectories of individual neural cell types in many neural systems including the neural retina. Further understanding of the formation of neural cell diversity requires knowledge about how the epigenetic landscape shifts along individual cell lineages and how key transcription factors regulate these changes. In this study, we dissect the changes in the epigenetic landscape during early retinal cell differentiation by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages. Using CUT&Tag, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2 and Isl1, including those dependent on Atoh7, and uncover the sequential and combinatorial interactions of these factors with the epigenetic landscape to control gene expression along individual retinal cell lineages such as retinal ganglion cells (RGCs). Our results reveal a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from multipotent retinal progenitor cells (RPCs).

Setd5, but not Setd2, is indispensable for retinal cell survival and proliferation.

Trimethylation of histone H3 at lysine 36 (H3K36me3) is associated with active transcription. We used mouse retinal explant cultures and shRNA to investigate the roles of Setd2 and Setd5, which encode H3K36me3 methyltransferases, in retinal development. We found that shSetd5 caused abnormal retinal structures and reduced rods and Müller cells, whereas shSetd2 did not cause any abnormalities. The mutant SETD5 lacking the SET domain failed to reverse the phenotypes observed in the shSetd5-expressing retinas, while SETD5S1257*, which does not interact with HDAC3 and PAF1 complexes, rescued proliferation, but not apoptosis, induced by shSetd5. Taken together, we found that Setd5, but not Setd2, is essential for sustaining retinal cell survival and proliferation, and the SET domain of SETD5 is pivotal for both functions.

Cephalopod retinal development shows vertebrate-like mechanisms of neurogenesis.

Coleoid cephalopods, including squid, cuttlefish, and octopus, have large and complex nervous systems and high-acuity, camera-type eyes. These traits are comparable only to features that are independently evolved in the vertebrate lineage. The size of animal nervous systems and the diversity of their constituent cell types is a result of the tight regulation of cellular proliferation and differentiation in development. Changes in the process of development during evolution that result in a diversity of neural cell types and variable nervous system size are not well understood. Here, we have pioneered live-imaging techniques and performed functional interrogation to show that the squid Doryteuthis pealeii utilizes mechanisms during retinal neurogenesis that are hallmarks of vertebrate processes. We find that retinal progenitor cells in the squid undergo nuclear migration until they exit the cell cycle. We identify retinal organization corresponding to progenitor, post-mitotic, and differentiated cells. Finally, we find that Notch signaling may regulate both retinal cell cycle and cell fate. Given the convergent evolution of elaborate visual systems in cephalopods and vertebrates, these results reveal common mechanisms that underlie the growth of highly proliferative neurogenic primordia. This work highlights mechanisms that may alter ontogenetic allometry and contribute to the evolution of complexity and growth in animal nervous systems.

The mitochondrial micropeptide Stmp1 promotes retinal cell differentiation.

During mammalian retinal development, the differentiation of multipotent progenitors depends on the coordinated action of a variety of intrinsic factors including non-coding RNAs (ncRNAs). To date, many small open reading frames have been identified in ncRNAs to encode micropeptides that function in diverse biological processes; however, it remains unclear whether they have a role in retinal development. Here we report that the 47-amino acid (AA) mitochondrial micropeptide Stmp1 encoded by the lncRNA 1810058I24Rik is involved in retinal differentiation. As the major protein product of 1810058I24Rik, Stmp1 promotes the differentiation of bipolar, amacrine and Müller cells as 1810058I24Rik does when overexpressed in neonatal murine retinas. Moreover, we have identified the 15-AA N-terminus of Stmp1 as its mitochondrion-targeting sequence as well as 5 conserved AA residues that affect protein stability and/or retinal cell differentiation. Together, our data reveal several novel characteristics of Stmp1 and uncover a role for Stmp1 in retinal cell differentiation perhaps through regulating mitochondrial function.

Modular strategy for development of the hierarchical visual network in mice.

Hierarchical and parallel networks are fundamental structures of the mammalian brain1-8. During development, lower- and higher-order thalamic nuclei and many cortical areas in the visual system form interareal connections and build hierarchical dorsal and ventral streams9-13. One hypothesis for the development of visual network wiring involves a sequential strategy wherein neural connections are sequentially formed alongside hierarchical structures from lower to higher areas14-17. However, this sequential strategy would be inefficient for building the entire visual network comprising numerous interareal connections. We show that neural pathways from the mouse retina to primary visual cortex (V1) or dorsal/ventral higher visual areas (HVAs) through lower- or higher-order thalamic nuclei form as parallel modules before corticocortical connections. Subsequently, corticocortical connections among V1 and HVAs emerge to combine these modules. Retina-derived activity propagating the initial parallel modules is necessary to establish retinotopic inter-module connections. Thus, the visual network develops in a modular manner involving initial establishment of parallel modules and their subsequent concatenation. Findings in this study raise the possibility that parallel modules from higher-order thalamic nuclei to HVAs act as templates for cortical ventral and dorsal streams and suggest that the brain has an efficient strategy for the development of a hierarchical network comprising numerous areas.

The Rx transcription factor is required for determination of the retinal lineage and regulates the timing of neuronal differentiation.

Understanding the molecular mechanisms leading to retinal development is of great interest for both basic scientific and clinical applications. Several signaling molecules and transcription factors involved in retinal development have been isolated and analyzed; however, determining the direct impact of the loss of a specific molecule is problematic, due to difficulties in identifying the corresponding cellular lineages in different individuals. Here, we conducted genome-wide expression analysis with embryonic stem (ES) cells devoid of the Rx gene, which encodes one of several homeobox transcription factors essential for retinal development. We performed three-dimensional differentiation of wild-type and mutant cells and compared their gene-expression profiles. The mutant tissue failed to differentiate into the retinal lineage and exhibited precocious expression of genes characteristic of neuronal cells. Together, these results suggest that Rx expression is an important biomarker of the retinal lineage and that it helps regulates appropriate differentiation stages.

[Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro].

OBJECTIVE: To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition. METHODS: SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for ß-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture. RESULTS: The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and ß-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05). CONCLUSION: In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.

Emergence of Direction-Selective Retinal Cell Types in Task-Optimized Deep Learning Models.

Convolutional neural networks (CNNs), a class of deep learning models, have experienced recent success in modeling sensory cortices and retinal circuits through optimizing performance on machine learning tasks, otherwise known as task optimization. Previous research has shown task-optimized CNNs to be capable of providing explanations as to why the retina efficiently encodes natural stimuli and how certain retinal cell types are involved in efficient encoding. In our work, we sought to use task-optimized CNNs as a means of explaining computational mechanisms responsible for motion-selective retinal circuits. We designed a biologically constrained CNN and optimized its performance on a motion-classification task. We drew inspiration from psychophysics, deep learning, and systems neuroscience literature to develop a toolbox of methods to reverse engineer the computational mechanisms learned in our model. Through reverse engineering our model, we proposed a computational mechanism in which direction-selective ganglion cells and starburst amacrine cells, both experimentally observed retinal cell types, emerge in our model to discriminate among moving stimuli. This emergence suggests that direction-selective circuits in the retina are ecologically designed to robustly discriminate among moving stimuli. Our results and methods also provide a framework for how to build more interpretable deep learning models and how to understand them.

NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation.

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Pericyte dysfunction and loss of interpericyte tunneling nanotubes promote neurovascular deficits in glaucoma.

Reduced blood flow and impaired neurovascular coupling are recognized features of glaucoma, the leading cause of irreversible blindness worldwide, but the mechanisms underlying these defects are unknown. Retinal pericytes regulate microcirculatory blood flow and coordinate neurovascular coupling through interpericyte tunneling nanotubes (IP-TNTs). Using two-photon microscope live imaging of the mouse retina, we found reduced capillary diameter and impaired blood flow at pericyte locations in eyes with high intraocular pressure, the most important risk factor to develop glaucoma. We show that IP-TNTs are structurally and functionally damaged by ocular hypertension, a response that disrupted light-evoked neurovascular coupling. Pericyte-specific inhibition of excessive Ca2+ influx rescued hemodynamic responses, protected IP-TNTs and neurovascular coupling, and enhanced retinal neuronal function as well as survival in glaucomatous retinas. Our study identifies pericytes and IP-TNTs as potential therapeutic targets to counter ocular pressure-related microvascular deficits, and provides preclinical proof of concept that strategies aimed to restore intrapericyte calcium homeostasis rescue autoregulatory blood flow and prevent neuronal dysfunction.