Txnip deletions and missense alleles prolong the survival of cones in a retinitis pigmentosa mouse model.

Retinitis pigmentosa (RP) is an inherited retinal disease in which there is a loss of cone-mediated daylight vision. As there are >100 disease genes, our goal is to preserve cone vision in a disease gene-agnostic manner. Previously we showed that overexpressing TXNIP, an α-arrestin protein, prolonged cone vision in RP mouse models, using an AAV to express it only in cones. Here, we expressed different alleles of Txnip in the retinal pigmented epithelium (RPE), a support layer for cones. Our goal was to learn more of TXNIP's structure-function relationships for cone survival, as well as determine the optimal cell type expression pattern for cone survival. The C-terminal half of TXNIP was found to be sufficient to remove GLUT1 from the cell surface, and improved RP cone survival, when expressed in the RPE, but not in cones. Knock-down of HSP90AB1, a TXNIP-interactor which regulates metabolism, improved the survival of cones alone and was additive for cone survival when combined with TXNIP. From these and other results, it is likely that TXNIP interacts with several proteins in the RPE to indirectly support cone survival, with some of these interactions different from those that lead to cone survival when expressed only in cones.

Txnip deletions and missense alleles prolong the survival of cones in a retinitis pigmentosa mouse model.

Retinitis pigmentosa (RP) is a prevalent inherited retinal degenerative disease worldwide, affecting 1 in 4,000 people. The disease is characterized by an initial loss of night vision followed by a loss of daylight and color vision. Many of the RP disease genes are expressed in the rod photoreceptors, the cell type that initiates dim light vision. Following loss of rods, the cone photoreceptors, which initiate daylight vision, also are affected and can die leading to total loss of vision. The reasons for loss of cone vision are not entirely clear, but appear to be due to loss of the rods. Previously we showed that overexpressing Txnip, an α-arrestin protein, in mouse models of RP using AAV gene therapy prolonged the survival of RP cones (Xue et al., 2021). At least part of the mechanism for cone survival was a switch in the fuel source, from glucose to lactate. In addition, the mitochondria of cones were both morphologically and functionally improved by delivery of Txnip. We have gone on to test several alleles of Txnip for the ability to prolong cone survival in rd1, a mouse model of RP. In addition, proteins that bind to Txnip and/or have homology to Txnip were tested. Five different deletion alleles of Txnip were expressed in cones or the retinal pigmented epithelium (RPE). Here we show that the C-terminal half of Txnip (149-397aa) is sufficient to remove GLUT1 from the RPE cell surface, and improved rd1 cone survival when expressed specifically in the RPE. Overexpressing Arrdc4, an α-arrestin that shares 60% similar protein sequence to Txnip, reduced rd1 cone survival. Reduction of the expression of HSP90AB1, a protein that interacts with Txnip and regulates metabolism, improved the survival of rd1 cones alone and was additive for cone survival when combined with Txnip. However, full length Txnip with a single amino acid change, C247S, as we tested in our original study, remains the most highly efficacious form of the gene for cone rescue. The above observations suggest that only a subset of the hypothesized and known activities of Txnip play a role in promoting RP cone survival, and that the activities of Txnip in the RPE differ from those in cone photoreceptors.

Thermal-plex: fluidic-free, rapid sequential multiplexed imaging with DNA-encoded thermal channels.

Multiplexed fluorescence imaging is typically limited to three- to five-plex on standard setups. Sequential imaging methods based on iterative labeling and imaging enable practical higher multiplexing, but generally require a complex fluidic setup with several rounds of slow buffer exchange (tens of minutes to an hour for each exchange step). We report the thermal-plex method, which removes complex and slow buffer exchange steps and provides fluidic-free, rapid sequential imaging. Thermal-plex uses simple DNA probes that are engineered to fluoresce sequentially when, and only when, activated with transient exposure to heating spikes at designated temperatures (thermal channels). Channel switching is fast (<30 s) and is achieved with a commercially available and affordable on-scope heating device. We demonstrate 15-plex RNA imaging (five thermal × three fluorescence channels) in fixed cells and retina tissues in less than 4 min, without using buffer exchange or fluidics. Thermal-plex introduces a new labeling method for efficient sequential multiplexed imaging.

High temporal frequency light response in mouse retina is mediated by ON and OFF bipolar cells and requires FAT3 signaling.

Vision is initiated by the reception of light by photoreceptors and subsequent processing via parallel retinal circuits. Proper circuit organization depends on the multi-functional tissue polarity protein FAT3, which is required for amacrine cell connectivity and retinal lamination. Here we investigated the retinal function of Fat3 mutant mice and found decreases in physiological and perceptual responses to high frequency flashes. These defects did not correlate with abnormal amacrine cell wiring, pointing instead to a role in bipolar cell subtypes that also express FAT3. Indeed, similar deficits were observed in mice lacking the bipolar cell glutamate receptors GRIK1 (OFF-bipolar cells) and GRM6 (ON-bipolar cells). Mechanistically, FAT3 binds to the synaptic protein PTPσ and is required to localize GRIK1 to OFF-cone bipolar cell synapses with cone photoreceptors. How FAT3 impacts ON-cone bipolar cell function at high temporal frequency remains to be uncovered. These findings expand the repertoire of FAT3's functions and reveal the importance of both ON- and OFF-bipolar cells for high frequency light response.

Gene Therapies for Retinitis Pigmentosa that Target Glucose Metabolism.

Retinitis pigmentosa is a blinding disease wherein rod photoreceptors are affected first, due to the expression of a disease gene, leading to the loss of dim light vision. In many cases, cones do not express the disease gene, yet they are also affected and eventually die, typically after most of the rods in their neighborhood have died. The cause of secondary cone death is unclear. Photoreceptors are one of the most energy-demanding cell types in the body and consume a high amount of glucose. At an early stage of degeneration, the cones appear to have a shortage of glucose to fuel their metabolism. This review focuses on gene therapy approaches that address this potential metabolic shortcoming.

Chromophore supply modulates cone function and survival in retinitis pigmentosa mouse models.

Retinitis pigmentosa (RP) is an ocular disease characterized by the loss of night vision, followed by the loss of daylight vision. Daylight vision is initiated in the retina by cone photoreceptors, which are gradually lost in RP, often as bystanders in a disease process that initiates in their neighboring rod photoreceptors. Using physiological assays, we investigated the timing of cone electroretinogram (ERG) decline in RP mouse models. A correlation between the time of loss of the cone ERG and the loss of rods was found. To investigate a potential role of the visual chromophore supply in this loss, mouse mutants with alterations in the regeneration of the retinal chromophore, 11-cis retinal, were examined. Reducing chromophore supply via mutations in Rlbp1 or Rpe65 resulted in greater cone function and survival in a RP mouse model. Conversely, overexpression of Rpe65 and Lrat, genes that can drive the regeneration of the chromophore, led to greater cone degeneration. These data suggest that abnormally high chromophore supply to cones upon the loss of rods is toxic to cones, and that a potential therapy in at least some forms of RP is to slow the turnover and/or reduce the level of visual chromophore in the retina.

A general approach for stabilizing nanobodies for intracellular expression.

Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli. These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.

We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.

Targeting Microglia to Treat Degenerative Eye Diseases.

Microglia have been implicated in many degenerative eye disorders, including retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, uveitis, and retinal detachment. While the exact roles of microglia in these conditions are still being discovered, evidence from animal models suggests that they can modulate the course of disease. In this review, we highlight current strategies to target microglia in the eye and their potential as treatments for both rare and common ocular disorders. These approaches include depleting microglia with chemicals or radiation, reprogramming microglia using homeostatic signals or other small molecules, and inhibiting the downstream effects of microglia such as by blocking cytokine activity or phagocytosis. Finally, we describe areas of future research needed to fully exploit the therapeutic value of microglia in eye diseases.

Retinoic acid signaling mediates peripheral cone photoreceptor survival in a mouse model of retina degeneration.

Retinitis Pigmentosa (RP) is a progressive, debilitating visual disorder caused by mutations in a diverse set of genes. In both humans with RP and mouse models of RP, rod photoreceptor dysfunction leads to loss of night vision, and is followed by secondary cone photoreceptor dysfunction and degeneration, leading to loss of daylight color vision. A strategy to prevent secondary cone death could provide a general RP therapy to preserve daylight color vision regardless of the underlying mutation. In mouse models of RP, cones in the peripheral retina survive long-term, despite complete rod loss. The mechanism for such peripheral cone survival had not been explored. Here, we found that active retinoic acid (RA) signaling in peripheral Muller glia is necessary for the abnormally long survival of these peripheral cones. RA depletion by conditional knockout of RA synthesis enzymes, or overexpression of an RA degradation enzyme, abrogated the extended survival of peripheral cones. Conversely, constitutive activation of RA signaling in the central retina promoted long-term cone survival. These results indicate that RA signaling mediates the prolonged peripheral cone survival in the rd1 mouse model of retinal degeneration, and provide a basis for a generic strategy for cone survival in the many diseases that lead to loss of cone-mediated vision.

Mouse Lines with Cre-Mediated Recombination in Retinal Amacrine Cells.

Amacrine cells (ACs) are the most diverse neuronal cell type in the vertebrate retina. Yet little is known about the contribution of ACs to visual processing and retinal disease. A major challenge in evaluating AC function is genetic accessibility. A classic tool of mouse genetics, Cre-mediated recombination, can provide such access. We have screened existing genetically-modified mouse strains and identified multiple candidates that express Cre-recombinase in subsets of retinal ACs. The Cre-expressing mice were crossed to fluorescent-reporter mice to assay Cre expression. In addition, a Cre-dependent fluorescent reporter plasmid was electroporated into the subretinal space of Cre strains. Herein, we report three mouse lines (Tac1::IRES-cre, Camk2a-cre, and Scx-cre) that express Cre recombinase in sub-populations of ACs. In two of these lines, recombination occurred in multiple AC types and a small number of other retinal cell types, while recombination in the Camk2a-cre line appears specific to a morphologically distinct AC. We anticipate that these characterized mouse lines will be valuable tools to the community of researchers who study retinal biology and disease.

Spatiotemporal patterns of neuronal subtype genesis suggest hierarchical development of retinal diversity.

How do neuronal subtypes emerge during development? Recent molecular studies have profiled existing neuronal diversity, but neuronal subtype genesis remains elusive. The 15 types of mouse retinal bipolar interneurons are characterized by distinct functions, morphologies, and transcriptional profiles. Here, we develop a comprehensive spatiotemporal map of bipolar subtype genesis in the murine retina. Combining multiplexed detection of 16 RNA markers with timed delivery of 5-ethynyl uridine (EdU) and bromodeoxyuridine (BrdU), we analyze more than 30,000 single cells in full retinal sections to classify all bipolar subtypes and their birthdates. We find that bipolar subtype birthdates are ordered and follow a centrifugal developmental axis. Spatial analysis reveals a striking wave pattern of bipolar subtype birthdates, and lineage analyses suggest clonal restriction on homotypic subtype production. These results inspire a hierarchical developmental model, with ordered subtype genesis within lineages. Our results provide insight into neuronal subtype development and establish a framework for studying subtype diversification.

Development and diversification of bipolar interneurons in the mammalian retina.

The bipolar interneurons of the mammalian retina have evolved as a diverse set of cells with distinct subtype characteristics, which reflect specialized contributions to visual circuitry. Fifteen subtypes of bipolar interneurons have been identified in the mouse retina, each with characteristic gene expression, morphology, and light responses. This review provides an overview of the developmental events that underlie the generation of the diverse bipolar cell class, summarizing the current knowledge of genetic programs that establish and maintain bipolar subtype fates, as well as the events that shape the final distribution of bipolar subtypes. With much left to be discovered, bipolar interneurons present an ideal model system for studying the interplay between cell-autonomous and non-cell-autonomous mechanisms that influence neuronal subtype development within the central nervous system.

Clinical Assessment and Validation of a Rapid and Sensitive SARS-CoV-2 Test Using Reverse Transcription Loop-Mediated Isothermal Amplification Without the Need for RNA Extraction.

BACKGROUND: Amid the enduring pandemic, there is an urgent need for expanded access to rapid, sensitive, and inexpensive coronavirus disease 2019 (COVID-19) testing worldwide without specialized equipment. We developed a simple test that uses colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect severe acute resrpiratory syndrome coronavirus 2 (SARS-CoV-2) in 40 minutes from sample collection to result. METHODS: We tested 135 nasopharyngeal specimens from patients evaluated for COVID-19 infection at Massachusetts General Hospital. Specimens were either added directly to RT-LAMP reactions, inactivated by a combined chemical and heat treatment step, or inactivated then purified with a silica particle-based concentration method. Amplification was performed with 2 SARS-CoV-2-specific primer sets and an internal specimen control; the resulting color change was visually interpreted. RESULTS: Direct RT-LAMP testing of unprocessed specimens could only reliably detect samples with abundant SARS-CoV-2 (>3 000 000 copies/mL), with sensitivities of 50% (95% CI, 28%-72%) and 59% (95% CI, 43%-73%) in samples collected in universal transport medium and saline, respectively, compared with quantitative polymerase chain reaction (qPCR). Adding an upfront RNase inactivation step markedly improved the limit of detection to at least 25 000 copies/mL, with 87.5% (95% CI, 72%-95%) sensitivity and 100% specificity (95% CI, 87%-100%). Using both inactivation and purification increased the assay sensitivity by 10-fold, achieving a limit of detection comparable to commercial real-time PCR-based diagnostics. CONCLUSIONS: By incorporating a fast and inexpensive sample preparation step, RT-LAMP accurately detects SARS-CoV-2 with limited equipment for about US$6 per sample, making this a potentially ideal assay to increase testing capacity, especially in resource-limited settings.

Augmentation of CD47/SIRPα signaling protects cones in genetic models of retinal degeneration.

Inherited retinal diseases, such as retinitis pigmentosa (RP), can be caused by thousands of different mutations, a small number of which have been successfully treated with gene replacement. However, this approach has yet to scale and may not be feasible in many cases, highlighting the need for interventions that could benefit more patients. Here, we found that microglial phagocytosis is upregulated during cone degeneration in RP, suggesting that expression of "don't-eat-me" signals such as CD47 might confer protection to cones. To test this, we delivered an adeno-associated viral (AAV) vector expressing CD47 on cones, which promoted cone survival in 3 mouse models of RP and preserved visual function. Cone rescue with CD47 required a known interacting protein, signal regulatory protein α (SIRPα), but not an alternative interacting protein, thrombospondin-1 (TSP1). Despite the correlation between increased microglial phagocytosis and cone death, microglia were dispensable for the prosurvival activity of CD47, suggesting that CD47 interacts with SIRPα on nonmicroglial cells to alleviate degeneration. These findings establish augmentation of CD47/SIRPα signaling as a potential treatment strategy for RP and possibly other forms of neurodegeneration.

AAV-Txnip prolongs cone survival and vision in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal disease affecting >20 million people worldwide. Loss of daylight vision typically occurs due to the dysfunction/loss of cone photoreceptors, the cell type that initiates our color and high-acuity vision. Currently, there is no effective treatment for RP, other than gene therapy for a limited number of specific disease genes. To develop a disease gene-agnostic therapy, we screened 20 genes for their ability to prolong cone photoreceptor survival in vivo. Here, we report an adeno-associated virus vector expressing Txnip, which prolongs the survival of cone photoreceptors and improves visual acuity in RP mouse models. A Txnip allele, C247S, which blocks the association of Txnip with thioredoxin, provides an even greater benefit. Additionally, the rescue effect of Txnip depends on lactate dehydrogenase b (Ldhb) and correlates with the presence of healthier mitochondria, suggesting that Txnip saves RP cones by enhancing their lactate catabolism.

Retinitis pigmentosa is an inherited eye disease affecting around one in every 4,000 people. It results from genetic defects in light sensitive cells of the retina, called photoreceptor cells, which line the back of the eye. Though vision loss can occur from birth, retinitis pigmentosa usually involves a gradual loss of vision, sometimes leading to blindness. Rod photoreceptors, which are responsible for vision in low light, are impacted first. The disease then affects cone photoreceptors, the cells that detect light during the day, providing both color and sharp vision. Around 100 mutated genes associated with retinitis pigmentosa have been identified, but only a handful of families with one of these mutant genes have been treated with a gene therapy specific for their mutated gene. There are currently no therapies available to treat the vast number of people with this disease. The mutations that cause retinitis pigmentosa directly affect the rod cells that detect dim light, leading to loss of night vision. There is also an indirect effect that causes cone photoreceptors to stop working and die. One theory to explain this two-step disease process relates to the fact that cone photoreceptors are very active cells, requiring a high level of energy, nutrients and oxygen. If surrounding rod cells die, cone photoreceptors may be deprived of some essential supplies, leading to cone cell death and daylight vision loss. To examine this theory, Xue et al. tested a new gene therapy designed to alleviate the potential shortfall in nutrients. The experiments used three different strains of mice that had the same genetic mutations as humans with retinitis pigmentosa. The gene therapy used a virus, called adeno-associated virus (AAV), to deliver 20 different genes to cone cells. Each of the 20 genes tested plays a different role in cells' processing of nutrients to provide energy. After administering the treatment, Xue et al. monitored the mice to see whether or not their vision was affected, and how cone cells responded. Only one of the 20 genes, Txnip, delivered using gene therapy, had a beneficial effect, prolonging cone cell survival in all three mouse strains. The mice that received Txnip also retained their ability to discern moving stripes on vision tests. Further investigations demonstrated that activating Txnip forced the cones to start using a molecule called lactate as an energy source, which could be more available to them than glucose, their usual fuel. These cells also had healthier mitochondria ­ the compartments inside cells that produce and manage energy supplies. This dual effect on fuel use and mitochondrial health is thought to be the basis for the extended cone survival and function. These experiments by Xue et al. have identified a good gene therapy candidate for treating retinitis pigmentosa independently of which genes are causing the disease. Further research will be required to test the safety of the gene therapy, and whether its beneficial effects translate to humans with retinitis pigmentosa, and potentially other diseases with unhealthy photoreceptors.

Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.

Nucleic acids are used in many therapeutic modalities, including gene therapy, but their ability to trigger host immune responses in vivo can lead to decreased safety and efficacy. In the case of adeno-associated viral (AAV) vectors, studies have shown that the genome of the vector activates Toll-like receptor 9 (TLR9), a pattern recognition receptor that senses foreign DNA. Here, we engineered AAV vectors to be intrinsically less immunogenic by incorporating short DNA oligonucleotides that antagonize TLR9 activation directly into the vector genome. The engineered vectors elicited markedly reduced innate immune and T cell responses and enhanced gene expression in clinically relevant mouse and pig models across different tissues, including liver, muscle, and retina. Subretinal administration of higher-dose AAV in pigs resulted in photoreceptor pathology with microglia and T cell infiltration. These adverse findings were avoided in the contralateral eyes of the same animals that were injected with the engineered vectors. However, intravitreal injection of higher-dose AAV in macaques, a more immunogenic route of administration, showed that the engineered vector delayed but did not prevent clinical uveitis, suggesting that other immune factors in addition to TLR9 may contribute to intraocular inflammation in this model. Our results demonstrate that linking specific immunomodulatory noncoding sequences to much longer therapeutic nucleic acids can "cloak" the vector from inducing unwanted immune responses in multiple, but not all, models. This "coupled immunomodulation" strategy may widen the therapeutic window for AAV therapies as well as other DNA-based gene transfer methods.

Nrf2 overexpression rescues the RPE in mouse models of retinitis pigmentosa.

Nrf2, a transcription factor that regulates the response to oxidative stress, has been shown to rescue cone photoreceptors and slow vision loss in mouse models of retinal degeneration (rd). The retinal pigment epithelium (RPE) is damaged in these models, but whether it also could be rescued by Nrf2 has not been previously examined. We used an adeno-associated virus (AAV) with an RPE-specific (Best1) promoter to overexpress Nrf2 in the RPE of rd mice. Control rd mice showed disruption of the regular array of the RPE, as well as loss of RPE cells. Cones were lost in circumscribed regions within the cone photoreceptor layer. Overexpression of Nrf2 specifically in the RPE was sufficient to rescue the RPE, as well as the disruptions in the cone photoreceptor layer. Electron microscopy showed compromised apical microvilli in control rd mice but showed preserved microvilli in Best1-Nrf2-treated mice. The rd mice treated with Best1-Nrf2 had slightly better visual acuity. Transcriptome profiling showed that Nrf2 upregulates multiple oxidative defense pathways, reversing declines seen in the glutathione pathway in control rd mice. In summary, Nrf2 overexpression in the RPE preserves RPE morphology and survival in rd mice, and it is a potential therapeutic for diseases involving RPE degeneration, including age-related macular degeneration (AMD).

In Situ Detection of Adeno-associated Viral Vector Genomes with SABER-FISH.

Gene therapy with recombinant adeno-associated viral (AAV) vectors is a promising modality for the treatment of a variety of human diseases. Nonetheless, there remain significant gaps in our understanding of AAV vector biology, due in part to the lack of robust methods to track AAV capsids and genomes. In this study, we describe a novel application of signal amplification by exchange reaction fluorescence in situ hybridization (SABER-FISH) that enabled the visualization and quantification of individual AAV genomes after vector administration in mice. These genomes could be seen in retinal cells within 3 h of subretinal AAV delivery, were roughly full length, and correlated with vector expression in both photoreceptors and the retinal pigment epithelium. SABER-FISH readily detected AAV genomes in the liver and muscle following retro-orbital and intramuscular AAV injections, respectively, demonstrating its utility in different tissues. Using SABER-FISH, we also found that retinal microglia, a cell type deemed refractory to AAV transduction, are in fact efficiently infected by multiple AAV serotypes, but appear to degrade AAV genomes prior to nuclear localization. Our findings show that SABER-FISH can be used to visualize AAV genomes in situ, allowing for studies of AAV vector biology and the tracking of transduced cells following vector administration.

Cell type- and stage-specific expression of Otx2 is regulated by multiple transcription factors and cis-regulatory modules in the retina.

Transcription factors (TFs) are often used repeatedly during development and homeostasis to control distinct processes in the same and/or different cellular contexts. Considering the limited number of TFs in the genome and the tremendous number of events that need to be regulated, re-use of TFs is necessary. We analyzed how the expression of the homeobox TF, orthodenticle homeobox 2 (Otx2), is regulated in a cell type- and stage-specific manner during development in the mouse retina. We identified seven Otx2 cis-regulatory modules (CRMs), among which the O5, O7 and O9 CRMs mark three distinct cellular contexts of Otx2 expression. We discovered that Otx2, Crx and Sox2, which are well-known TFs regulating retinal development, bind to and activate the O5, O7 or O9 CRMs, respectively. The chromatin status of these three CRMs was found to be distinct in vivo in different retinal cell types and at different stages. We conclude that retinal cells use a cohort of TFs with different expression patterns and multiple CRMs with different chromatin configurations to regulate the expression of Otx2 precisely.