Müller cells are a crucial retinal cell type involved in multiple regulatory processes and functions that are essential for retinal health and functionality. Acting as structural and functional support for retinal neurons and photoreceptors, Müller cells produce growth factors, regulate ion and fluid homeostasis, and facilitate neuronal signaling. They play a pivotal role in retinal morphogenesis and cell differentiation, significantly contributing to macular development. Due to their radial morphology and unique cytoskeletal organization, Müller cells act as optical fibers, efficiently channeling photons directly to the photoreceptors. In response to retinal damage, Müller cells undergo specific gene expression and functional changes that serve as a first line of defense for neurons, but can also lead to unwarranted cell dysfunction, contributing to cell death and neurodegeneration. In some species, Müller cells can reactivate their developmental program, promoting retinal regeneration and plasticity-a remarkable ability that holds promising therapeutic potential if harnessed in mammals. The crucial and multifaceted roles of Müller cells-that we propose to collectively call "Müller cells trophism"-highlight the necessity of maintaining their functionality. Dysfunction of Müller cells, termed "Müller cells pathology," has been associated with a plethora of retinal diseases, including age-related macular degeneration, diabetic retinopathy, vitreomacular disorders, macular telangiectasia, and inherited retinal dystrophies. In this review, we outline how even subtle disruptions in Müller cells trophism can drive the pathological cascade of Müller cells pathology, emphasizing the need for targeted therapies to preserve retinal health and prevent disease progression.

Retinal Muller 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 Muller glia, induction of neurogenesis in wild-type glia has thus far proven elusive. Here, we report that viral-mediated overexpression of the pluripotency factor Oct4 (Pou5f1) induces transdifferentiation of mouse Muller glia into bipolar neurons, and synergistically stimulates glial-derived neurogenesis in parallel with Notch loss of function. Single cell multiomic analysis shows that Oct4 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 Oct4 induces neurogenic competence in retinal Muller glia, identifying mechanisms that could be used in cell-based therapies for treating retinal dystrophies.

Small molecules like PLX5622 for microglia depletion and Tamoxifen for inducible Cre recombination are commonly used in mouse research. Traditional application methods, such as chow or oral gavage and injections, have limitations, including uncontrolled dosage and risk of injury. To address this issue, we have developed an alternative oral drug delivery system using a gel-based rodent maintenance diet that allows for controlled consumption and adjustment of dosage and is suitable for water-insoluble small molecules. We tested DietGel® 93M (93M) infused with PLX5622 (0.8 mg/g and 2.0 mg/g) in the Cx3cr1 gfp/+ retinal microglia reporter mouse and Tamoxifen-infused 93M (0.3125 mg/g) in the Rlbp1-CreERT2 ;Rosa ai14 mouse with an inducible tdTomato reporter in retinal Müller glia. Mice were single-caged and received daily batches of PLX5622-infused 93M over 14 days or Tamoxifen-infused 93M for one or three days followed by a 14-day observation period. Longitudinal scanning laser ophthalmoscopy in vivo and fixed tissue imaging were used to track GFP and tdTomato expression. Following evaluation of a suitable 93M consumption rate (g/d) to sustain body weight, the PLX5622-93M diet at both concentrations showed a 94% microglia depletion rate at 3 days and >99% after one and two weeks. The Tamoxifen-93M diet confirmed suitability for inducible Cre recombination, with significant treatment-time dependent efficacy and a positive correlation between total Tamoxifen dose and tdTomato expression. This study demonstrates that a diet gel-based drug delivery system offers a controllable and less invasive alternative to current drug application methods for PLX5622 and Tamoxifen.

Retinal diseases often lead to degeneration of specific retinal cell types with currently limited therapeutic options to replace the lost neurons. Previous studies have reported that overexpression of ASCL1 or combinations of proneural factors in Müller glia (MG) induce regeneration of functional neurons in the adult mouse retina. Recently, we applied the same strategy in dissociated cultures of fetal human MG and although we stimulated neurogenesis from MG, our effect in 2D cultures was modest and our analysis of newborn neurons was limited. In this study, we aimed to improve our MG reprogramming strategy in a more intact retinal environment. For this purpose, we used an in vitro culture system of human fetal retinal tissue and adult human postmortem retina. To stimulate reprogramming, we used lentiviral vectors to deliver constructs with a glial-specific promoter (HES1) driving ASCL1 alone or in combination with additional developmental transcription factors (TFs) such as ATOH1 and NEUROD1. Combining IHC, scRNA-seq, and electrophysiology, we show that human MG can generate new neurons even in adults. This work constitutes a key step toward a future clinical application of this regenerative medicine approach for retinal degenerative disorders.

Retinal degenerative diseases including age-related macular degeneration and glaucoma are estimated to currently affect more than 14 million people in the United States, with an increased prevalence of retinal degenerations in aged individuals. An expanding aged population who are living longer forecasts an increased prevalence and economic burden of visual impairments. Improvements to visual health and treatment paradigms for progressive retinal degenerations slow vision loss. However, current treatments fail to remedy the root cause of visual impairments caused by retinal degenerations-loss of retinal neurons. Stimulation of retinal regeneration from endogenous cellular sources presents an exciting treatment avenue for replacement of lost retinal cells. In multiple species including zebrafish and Xenopus, Müller glial cells maintain a highly efficient regenerative ability to reconstitute lost cells throughout the organism's lifespan, highlighting potential therapeutic avenues for stimulation of retinal regeneration in humans. Here, we describe how the application of single-cell RNA-sequencing (scRNA-seq) has enhanced our understanding of Müller glial cell-derived retinal regeneration, including the characterization of gene regulatory networks that facilitate/inhibit regenerative responses. Additionally, we provide a validated experimental framework for cellular preparation of mouse retinal cells as input into scRNA-seq experiments, including insights into experimental design and analyses of resulting data.

We summarize recent findings in different animal models regarding the different cell-signaling pathways and gene networks that influence the reprogramming of Müller glia into proliferating, neurogenic progenitor cells in the retina. Not surprisingly, most of the cell-signaling pathways that guide the proliferation and differentiation of embryonic retinal progenitors also influence the ability of Müller glia to become proliferating Müller glia-derived progenitor cells (MGPCs). Further, the neuronal differentiation of MGPC progeny is potently inhibited by networks of neurogenesis-suppressing genes in chick and mouse models but occurs freely in zebrafish. There are important differences between the model systems, particularly pro-inflammatory signals that are active in mature Müller glia in damaged rodent and chick retinas, but less so in fish retinas. These pro-inflammatory signals are required to initiate the process of reprogramming, but if sustained suppress the potential of Müller glia to become neurogenic MGPCs. Further, there are important differences in how activated Müller glia up- or downregulate pro-glial transcription factors in the different model systems. We review recent findings regarding regulatory cell signaling and gene networks that influence the activation of Müller glia and the transition of these glia into proliferating progenitor cells with neurogenic potential in fish, chick, and mouse model systems.

It is advantageous to culture the ex vivo retina and other tissues at the air-liquid interface to allow for more efficient gas exchange. However, gene delivery to these cultures can be challenging. Electroporation is a fast and robust method of gene delivery, but typically requires submergence in liquid buffer for electrical current flow. We have developed a submergence-free electroporation technique that incorporates an agarose hydrogel disk between the positive electrode and retina. Inner retinal neurons and Müller glia are transfected with increased propensity toward Müller glia transfection after extended time in culture. We also observed an increase in BrdU incorporation in Müller glia following electrical stimulation, and variation in detection of transfected cells from expression vectors with different promoters. This method advances our ability to use ex vivo retinal tissue for genetic studies and should be adaptable for other tissues cultured at an air-liquid interface.

Repairing the adult mammalian brain represents one of the greatest clinical challenges in medicine. Injury to the adult brain often results in substantial loss of neural tissue and permanent functional impairment. In contrast with the adult, during development, the mammalian brain exhibits a remarkable capacity to replace lost cells. A plethora of cell-intrinsic and extrinsic factors regulate the age-dependent loss of regenerative potential in the brain. As the developmental window closes, neural stem cells undergo epigenetic changes, limiting their proliferation and differentiation capacities, whereas, changes in the brain microenvironment pose additional challenges opposing regeneration, including inflammation and gliosis. Therefore, studying the regenerative mechanisms during development and identifying what impairs them with age may provide key insights into how to stimulate regeneration in the brain. Here, we will discuss how the mammalian brain engages regenerative mechanisms upon injury or neuron loss. Moreover, we will describe the age-dependent changes that affect these processes. We will conclude by discussing potential therapeutic approaches to overcome the age-dependent regenerative decline and stimulate regeneration.

Retinal degeneration in mammals causes permanent loss of vision, due to an inability to regenerate naturally. Some non-mammalian vertebrates show robust regeneration, via Muller glia (MG). We have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. With the goal of improving this in mammals, we designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single-nucleus RNA-seq conditions into a single experiment. We used this technology to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease.

Retinal neurodegeneration (RN), an early marker of diabetic retinopathy (DR), is closely associated with Müller glia cells (MGs) in diabetic subjects. MGs play a pivotal role in maintaining retinal homeostasis, integrity, and metabolic support and respond to diabetic stress. In lower vertebrates, MGs have a strong regenerative response and can completely repair the retina after injuries. However, this ability diminishes as organisms become more complex. The aim of this study was to investigate the gliotic response and reprogramming potential of the human Müller cell line MIO-M1 cultured in normoglycemic (5 mM glucose, NG) and hyperglycemic (25 mM glucose, HG) conditions and then exposed to sustained high-glucose and glucose fluctuation (GF) treatments to mimic the human diabetic conditions. The results showed that NG MIO-M1 cells exhibited a dynamic activation to sustained high-glucose and GF treatments by increasing GFAP and Vimentin expression together, indicative of gliotic response. Increased expression of SHH and SOX2 were also observed, foreshadowing reprogramming potential. Conversely, HG MIO-M1 cells showed increased levels of the indexes reported above and adaptation/desensitization to sustained high-glucose and GF treatments. These findings indicate that MIO-M1 cells exhibit a differential response under various glucose treatments, which is dependent on the metabolic environment. The in vitro model used in this study, based on a well-established cell line, enables the exploration of how these responses occur in a controlled, reproducible system and the identification of strategies to promote neurogenesis over neurodegeneration. These findings contribute to the understanding of MGs responses under diabetic conditions, which may have implications for future therapeutic approaches to diabetes-associated retinal neurodegeneration.

Inflammation and microglia appear to be key factors influencing the outcome of retinal regeneration following acute retinal damage. Despite such findings, direct connection of microglia-specific inflammatory factors as drivers of regenerative responses in the retina are still not defined, and intracellular pathways activated to stimulate such signals from microglia are currently unknown. We became interested in MyD88 regulation in microglia because transcriptomic datasets suggest myd88 could be regulated temporally in zebrafish microglia responding to damage in the central nervous system. MyD88 is an intracellular molecular adaptor that initiates signaling cascades downstream of several innate immune receptors, and probably most well-known for inducing gene expression of pro-inflammatory factors. Using zebrafish, which spontaneously regenerate retinal neurons after acute retinal damage, we studied the effects of overactivation of MyD88 signaling in microglia and macrophages on the Müller glia-mediated regenerative response. Our results indicate that increased MyD88 signaling in microglia/macrophages impacts the initial response of Müller glia entering a regenerative response after acute, neurotoxin-induced retinal damage to inner retinal neurons. In addition, increased MyD88 signaling in microglia/macrophages resulted in reduced survival of inner retinal neurons in regenerated retinas. This work supports the idea that temporal control of inflammatory signaling is a key component in the production of MG-derived progenitors yet further indicates that such control is important for differentiation and survival of regenerated neurons.

Retinal degeneration (RD) is a leading cause of blindness worldwide and includes conditions such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD). These diseases result in the permanent loss of vision due to the progressive and irreversible degeneration of retinal cells, including photoreceptors (PR) and the retinal pigment epithelium (RPE). The adult human retina has limited abilities to regenerate and repair itself, making it challenging to achieve complete self-replenishment and functional repair of retinal cells. Currently, there is no effective clinical treatment for RD. Stem cell therapy, which involves transplanting exogenous stem cells such as retinal progenitor cells (RPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), or activating endogenous stem cells like Müller Glia (MG) cells, holds great promise for regenerating and repairing retinal cells in the treatment of RD. Several preclinical and clinical studies have shown the potential of stem cell-based therapies for RD. However, the clinical translation of these therapies for the reconstruction of substantial vision still faces significant challenges. This review provides a comprehensive overview of stem/progenitor cell-based therapy strategies for RD, summarizes recent advances in preclinical studies and clinical trials, and highlights the major challenges in using stem/progenitor cell-based therapies for RD.

Neuronal injury, aging, and cerebrovascular and neurodegenerative diseases such as cerebral infarction, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis, and Huntington's disease are characterized by significant neuronal loss. Unfortunately, the neurons of most mammals including humans do not possess the ability to self-regenerate. Replenishment of lost neurons becomes an appealing therapeutic strategy to reverse the disease phenotype. Transplantation of pluripotent neural stem cells can supplement the missing neurons in the brain, but it carries the risk of causing gene mutation, tumorigenesis, severe inflammation, and obstructive hydrocephalus induced by brain edema. Conversion of neural or non-neural lineage cells into functional neurons is a promising strategy for the diseases involving neuron loss, which may overcome the above-mentioned disadvantages of neural stem cell therapy. Thus far, many strategies to transform astrocytes, fibroblasts, microglia, Müller glia, NG2 cells, and other glial cells to mature and functional neurons, or for the conversion between neuronal subtypes have been developed through the regulation of transcription factors, polypyrimidine tract binding protein 1 (PTBP1), and small chemical molecules or are based on a combination of several factors and the location in the central nervous system. However, some recent papers did not obtain expected results, and discrepancies exist. Therefore, in this review, we discuss the history of neuronal transdifferentiation, summarize the strategies for neuronal replenishment and conversion from glia, especially astrocytes, and point out that biosafety, new strategies, and the accurate origin of the truly converted neurons in vivo should be focused upon in future studies. It also arises the attention of replenishing the lost neurons from glia by gene therapies such as up-regulation of some transcription factors or down-regulation of PTBP1 or drug interference therapies.

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are widely expressed by many types of retinal neurons and are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin and FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases acting through retinal neurons and MG "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

In this review, we explore the connections between developmental embryology and axonal regeneration. Genes that regulate embryogenesis and central nervous system (CNS) development are discussed for their therapeutic potential to induce axonal and cellular regeneration in adult tissues after neuronal injury. Despite substantial differences in the tissue environment in the developing CNS compared with the injured CNS, recent studies have identified multiple molecular pathways that promote axonal growth in both scenarios. We describe various molecular cues and signaling pathways involved in neural development, with an emphasis on the versatile Wnt signaling pathway. We discuss the capacity of developmental factors to initiate axonal regrowth in adult neural tissue within the challenging environment of the injured CNS. Our discussion explores the roles of Wnt signaling and also examines the potential of other embryonic genes including Pax, BMP, Ephrin, SOX, CNTF, PTEN, mTOR and STAT3 to contribute to axonal regeneration in various CNS injury model systems, including spinal cord and optic crush injuries in mice, Xenopus and zebrafish. Additionally, we describe potential contributions of Müller glia redifferentiation to neuronal regeneration after injury. Therefore, this review provides a comprehensive summary of the state of the field, and highlights promising research directions for the potential therapeutic applications of specific embryologic molecular pathways in axonal regeneration in adults.

Retinal degeneration in mammals causes permanent loss of vision, due to an inability to regenerate naturally. Some non-mammalian vertebrates show robust regeneration, via Muller glia (MG). We have recently made significant progress in stimulating adult mouse MG to regenerate functional neurons by transgenic expression of the proneural transcription factor Ascl1. While these results showed that MG can serve as an endogenous source of neuronal replacement, the efficacy of this process is limited. With the goal of improving this in mammals, we designed a small molecule screen using sci-Plex, a method to multiplex up to thousands of single nucleus RNA-seq conditions into a single experiment. We used this technology to screen a library of 92 compounds, identified, and validated two that promote neurogenesis in vivo. Our results demonstrate that high-throughput single-cell molecular profiling can substantially improve the discovery process for molecules and pathways that can stimulate neural regeneration and further demonstrate the potential for this approach to restore vision in patients with retinal disease.

Aging causes numerous age-related diseases, leading the human species to death. Nevertheless, rejuvenating strategies based on cell epigenetic modifications are a possible approach to counteract disease progression while getting old. Cell reprogramming of adult somatic cells toward pluripotency ought to be a promising tool for age-related diseases. However, researchers do not have control over this process as cells lose their fate, and cause potential cancerous cells or unexpected cell phenotypes. Direct and partial reprogramming were introduced in recent years with distinctive applications. Although direct reprogramming makes cells lose their identity, it has various applications in regeneration medicine. Temporary and regulated in vivo overexpression of Yamanaka factors has been shown in several experimental contexts to be achievable and is used to rejuvenate mice models. This regeneration can be accomplished by altering the epigenetic adult cell signature to the signature of a younger cell. The greatest advantage of partial reprogramming is that this method does not allow cells to lose their identity when they are resetting their epigenetic clock. It is a regimen of short-term Oct3/4, Sox2, Klf4, and c-Myc expression in vivo that prevents full reprogramming to the pluripotent state and avoids both tumorigenesis and the presence of unwanted undifferentiated cells. We know that many neurological age-related diseases, such as Alzheimer's disease, stroke, dementia, and Parkinson's disease, are the main cause of death in the last decades of life. Therefore, scientists have a special tendency regarding neuroregeneration methods to increase human life expectancy.

The retina is the sensory tissue responsible for the first stages of visual processing, with a conserved anatomy and functional architecture among vertebrates. To date, retinal eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, glaucoma, and others, affect nearly 170 million people worldwide, resulting in vision loss and blindness. To tackle retinal disorders, the developing retina has been explored as a versatile model to study intercellular signaling, as it presents a broad neurochemical repertoire that has been approached in the last decades in terms of signaling and diseases. Retina, dissociated and arranged as typical cultures, as mixed or neuron- and glia-enriched, and/or organized as neurospheres and/or as organoids, are valuable to understand both neuronal and glial compartments, which have contributed to revealing roles and mechanisms between transmitter systems as well as antioxidants, trophic factors, and extracellular matrix proteins. Overall, contributions in understanding neurogenesis, tissue development, differentiation, connectivity, plasticity, and cell death are widely described. A complete access to the genome of several vertebrates, as well as the recent transcriptome at the single cell level at different stages of development, also anticipates future advances in providing cues to target blinding diseases or retinal dysfunctions.

Retinal Müller glia have a capacity to regenerate neurons in lower vertebrates like zebrafish, but such ability is extremely limited in mammals. In zebrafish, Müller glia proliferate after injury, which promotes their neurogenic reprogramming while inhibiting reactive gliosis. In mammals, however, how the cell cycle affects the fate of Müller glia after injury remains unclear. Here, we focused on the expression of proneural transcription factors, Ngn2 and Ascl1, and a gliosis marker glial fibrillary acidic protein (GFAP) in rat Müller glia after N-methyl-N-nitrosourea (MNU)-induced photoreceptor injury and analyzed the role of Müller glia proliferation in the regulation of their expression using retinal explant cultures. Thymidine-induced G1/S arrest of Müller glia proliferation significantly hampered the expression of Ascl1, Ngn2, and GFAP, and release from the arrest induced their upregulation. The migration of Müller glia nuclei into the outer nuclear layer was also shown to be cell cycle-dependent. These data suggest that, unlike the situation in zebrafish, cell cycle progression of Müller glia in mammals promotes both neurogenic reprogramming and reactive gliosis, which may be one of the mechanisms underlying the limited regenerative capacity of the mammalian retina.

Müller cells play an integral role in the development, maintenance, and photopic signal transmission of the retina. While lower vertebrate Müller cells can differentiate into various types of retinal neurons to support retinal repair following damage, there is limited neurogenic potential of mammalian Müller cells. Therefore, it is of great interest to harness the neurogenic potential of mammalian Müller cells to achieve self-repair of the retina. While multiple studies have endeavored to induce neuronal differentiation and proliferation of mammalian Müller cells under defined conditions, the efficiency and feasibility of these methods often fall short, rendering them inadequate for the requisites of retinal repair. As the mechanisms and methodologies of Müller cell reprogramming have been extensively explored, a summary of the reprogramming process of unlocking the neurogenic potential of Müller cells can provide insight into Müller cell fate development and facilitate their therapeutic use in retinal repair. In this review, we comprehensively summarize the progress in reprogramming mammalian Müller cells and discuss strategies for optimizing methods and enhancing efficiency based on the mechanisms of fate regulation.

Different kinase-dependent cell signaling pathways are known to play important roles in glia-mediated neuroprotection and reprogramming of Müller glia (MG) into Müller glia-derived progenitor cells (MGPCs) in the retina. However, very little is known about the phosphatases that regulate kinase-dependent signaling in MG. Using single-cell RNA-sequencing (scRNA-seq) databases, we investigated patterns of expression of Dual Specificity Phosphatases (DUSP1/6) and other protein phosphatases in normal and damaged chick retinas. We found that DUSP1, DUSP6, PPP3CB, PPP3R1 and PPPM1A/B/D/E/G are dynamically expressed by MG and MGPCs in retinas during the process of reprogramming. We find that inhibition of DUSP1/6 and PP2C phosphatases enhances the formation of proliferating MGPCs in damaged retinas and in retinas treated with insulin in FGF2 in the absence of damage. By contrast, inhibition of PP2B phosphatases suppressed the formation of proliferating MGPCs, but increased numbers of proliferating MGPCs in undamaged retinas treated with insulin and FGF2. In damaged retinas, inhibition of DUSP1/6 increased levels of pERK1/2 and cFos in MG whereas inhibition of PP2B's decreased levels of pStat3 and pS6 in MG. Analyses of scRNA-seq libraries identified numerous differentially activated gene modules in MG in damaged retinas versus MG in retinas treated with insulin+FGF2 suggesting significant differences in kinase-dependent signaling pathways that converge on the formation of MGPCs. Inhibition of phosphatases had no significant effects upon numbers of dying cells in damaged retinas. We conclude that the activity of different protein phosphatases "fine-tune" the cell signaling responses of MG in damaged retinas and during the reprogramming of MG into MGPCs.

In mammals, loss of retinal cells due to disease or trauma is an irreversible process that can lead to blindness. Interestingly, regeneration of retinal neurons is a well established process in some non-mammalian vertebrates and is driven by the Müller glia (MG), which are able to re-enter the cell cycle and reprogram into neurogenic progenitors upon retinal injury or disease. Progress has been made to restore this mechanism in mammals to promote retinal regeneration: MG can be stimulated to generate new neurons in vivo in the adult mouse retina after the over-expression of the pro-neural transcription factor Ascl1. In this study, we applied the same strategy to reprogram human MG derived from fetal retina and retinal organoids into neurons. Combining single cell RNA sequencing, single cell ATAC sequencing, immunofluorescence, and electrophysiology we demonstrate that human MG can be reprogrammed into neurogenic cells in vitro.

Significance: Retinal neurons are vulnerable to disease and injury, which can result in neuronal death and degeneration leading to irreversible vision loss. The human retina does not regenerate to replace neurons lost to disease or injury. However, cells within the retina of other animals are capable of regenerating neurons, and homologous cells within the mammalian retina could potentially be prompted to do the same. Activating evolutionarily silenced intrinsic regenerative capacity of the mammalian retina could slow, or even reverse, vision loss, leading to an improved quality of life for millions of people. Recent Advances: During development, neurons in the retina are generated progressively by retinal progenitor cells, with distinct neuron types born over developmental time. Many genes function in this process to specify the identity of newly generated neuron types, and these appropriate states of gene expression inform recent regenerative work. When regeneration is initiated in other vertebrates, including birds and fish, specific signaling pathways control the efficiency of regeneration, and these conserved pathways are likely to be important in mammals as well. Critical Issues: Using insights from development and from other animals, limited regeneration from intrinsic cell types has been demonstrated in the mammalian retina, but it is able only to generate a subset of partially differentiated retinal neuron types. Future Directions: Future studies should aim at increasing the efficiency of regeneration, activating regeneration in a targeted fashion across the retina, and improving the ability to generate specific types of retinal neurons to replace those lost to disease or injury. Antioxid. Redox Signal. 39, 1039-1052.

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.

The zebrafish retina possesses tremendous regenerative potential. Müller glia underlie retinal regeneration through their ability to reprogram and generate multipotent neuronal progenitors that re-differentiate into lost neurons. Many factors required for Müller glia reprogramming and proliferation have been identified; however, we know little about the epigenetic and transcriptional regulation of these genes during regeneration. Here, we determined whether transcriptional regulation by members of the Bromodomain (Brd) family is required for Müller glia-dependent retinal regeneration. Our data demonstrate that three brd genes were expressed in Müller glia upon injury. brd2a and brd2b were expressed in all Müller glia and brd4 was expressed only in reprogramming Müller glia. Utilizing (+)-JQ1, a pharmacological inhibitor of Brd function, we demonstrate that transcriptional regulation by Brds plays a critical role in Müller glia reprogramming and regeneration. (+)-JQ1 treatment prevented cell cycle re-entry of Müller glia and the generation of neurogenic progenitors. Modulating the (+)-JQ1 exposure window, we identified the first 48 h post-injury as the time-period during which Müller glia reprogramming occurs. (+)-JQ1 treatments after 48 h post-injury had no effect on the re-differentiation of UV cones, indicating that Brd function is required only for Müller glia reprogramming and not subsequent specification/differentiation events. Brd inhibition also prevented the expression of reprogramming genes like ascl1a and lepb in Müller glia, but not effector genes like mmp9, nor did it affect microglial recruitment after injury. These results demonstrate that transcriptional regulation by Brds plays a critical role during Müller glia-dependent retinal regeneration in zebrafish.

Recent studies have demonstrated the impact of pro-inflammatory signaling and reactive microglia/macrophages on the formation of Müller glial-derived progenitor cells (MGPCs) in the retina. In chick retina, ablation of microglia/macrophages prevents the formation of MGPCs. Analyses of single-cell RNA-sequencing chick retinal libraries revealed that quiescent and activated microglia/macrophages have a significant impact upon the transcriptomic profile of Müller glia (MG). In damaged monocyte-depleted retinas, MG fail to upregulate genes related to different cell signaling pathways, including those related to Wnt, heparin-binding epidermal growth factor (HBEGF), fibroblast growth factor (FGF) and retinoic acid receptors. Inhibition of GSK3β, to simulate Wnt signaling, failed to rescue the deficit in MGPC formation, whereas application of HBEGF or FGF2 completely rescued the formation of MGPCs in monocyte-depleted retinas. Inhibition of Smad3 or activation of retinoic acid receptors partially rescued the formation of MGPCs in monocyte-depleted retinas. We conclude that signals produced by reactive microglia/macrophages in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFβ/Smad3 to promote the reprogramming of MG into proliferating MGPCs.

The potential for basic research to uncover the inner workings of regenerative processes and produce meaningful medical therapies has inspired scientists, clinicians, and patients for hundreds of years. Decades of studies using a handful of highly regenerative model organisms have significantly advanced our knowledge of key cell types and molecular pathways involved in regeneration. However, many questions remain about how regenerative processes unfold in regeneration-competent species, how they are curtailed in non-regenerative organisms, and how they might be induced (or restored) in humans. Recent technological advances in genomics, molecular biology, computer science, bioengineering, and stem cell research hold promise to collectively provide new experimental evidence for how different organisms accomplish the process of regeneration. In theory, this new evidence should inform the design of new clinical approaches for regenerative medicine. A deeper understanding of how tissues and organs regenerate will also undoubtedly impact many adjacent scientific fields. To best apply and adapt these new technologies in ways that break long-standing barriers and answer critical questions about regeneration, we must combine the deep knowledge of developmental and evolutionary biologists with the hard-earned expertise of scientists in mechanistic and technical fields. To this end, this perspective is based on conversations from a workshop we organized at the Banbury Center, during which a diverse cross-section of the regeneration research community and experts in various technologies discussed enduring questions in regenerative biology. Here, we share the questions this group identified as significant and unanswered, i.e., known unknowns. We also describe the obstacles limiting our progress in answering these questions and how expanding the number and diversity of organisms used in regeneration research is essential for deepening our understanding of regenerative capacity. Finally, we propose that investigating these problems collaboratively across a diverse network of researchers has the potential to advance our field and produce unexpected insights into important questions in related areas of biology and medicine.

Damage to light-sensing photoreceptors (PRs) occurs in highly prevalent retinal diseases. As humans cannot regenerate new PRs, these diseases often lead to irreversible blindness. Intriguingly, animals, such as the zebrafish, can regenerate PRs efficiently and restore functional vision. Upon injury, mature Müller glia (MG) undergo reprogramming to adopt a stem cell-like state. This process is similar to cellular dedifferentiation, and results in the generation of progenitor cells, which, in turn, proliferate and differentiate to replace lost retinal neurons. In this study, we tested whether factors involved in dedifferentiation of Drosophila CNS are implicated in the regenerative response in the zebrafish retina. We found that hairy-related 6 (her6) negatively regulates of PR production by regulating the rate of cell divisions in the MG-derived progenitors. prospero homeobox 1a (prox1a) is expressed in differentiated PRs and may promote PR differentiation through phase separation. Interestingly, upon Her6 downregulation, Prox1a is precociously upregulated in the PRs, to promote PR differentiation; conversely, loss of Prox1a also induces a downregulation of Her6. Together, we identified two novel candidates of PR regeneration that cross regulate each other; these may be exploited to promote human retinal regeneration and vision recovery.

The neural retina of mammals, like most of the rest of the central nervous system, does not regenerate new neurons after they are lost through damage or disease. The ability of nonmammalian vertebrates, like fish and amphibians, is remarkable, and lessons learned over the last 20 years have revealed some of the mechanisms underlying this potential. This knowledge has recently been applied to mammals to develop methods that can stimulate regeneration in mice. In this review, we highlight the progress in this area, and propose a "wish list" of how the clinical implementation of regenerative strategies could be applicable to various human retinal diseases.

Retinal diseases characterized with irreversible loss of retinal nerve cells, such as optic atrophy and retinal degeneration, are the main causes of blindness. Current treatments for these diseases are very limited. An emerging treatment strategy is to induce the reprogramming of Müller glial cells to generate new retinal nerve cells, which could potentially restore vision.

Müller glial cells are the predominant glial cells in retinae and play multiple roles to maintain retinal homeostasis. In lower vertebrates, such as in zebrafish, Müller glial cells can undergo cell reprogramming to regenerate new retinal neurons in response to various damage factors, while in mammals, this ability is limited. Interestingly, with proper treatments, Müller glial cells can display the potential for regeneration of retinal neurons in mammalian retinae. Recent studies have revealed that dozens of genetic and epigenetic regulators play a vital role in inducing the reprogramming of Müller glial cells in vivo. This review summarizes these critical regulators for Müller glial cell reprogramming and highlights their differences between zebrafish and mammals.

A number of factors have been identified as the important regulators in Müller glial cell reprogramming. The early response of Müller glial cells upon acute retinal injury, such as the regulation in the exit from quiescent state, the initiation of reactive gliosis, and the re-entry of cell cycle of Müller glial cells, displays significant difference between mouse and zebrafish, which may be mediated by the diverse regulation of Notch and TGFβ (transforming growth factor-β) isoforms and different chromatin accessibility.

Loss of neurons in the neural retina is a leading cause of vision loss. While humans do not possess the capacity for retinal regeneration, zebrafish can achieve this through activation of resident Müller glia. Remarkably, despite the presence of Müller glia in humans and other mammalian vertebrates, these cells lack an intrinsic ability to contribute to regeneration. Upon activation, zebrafish Müller glia can adopt a stem cell-like state, undergo proliferation and generate new neurons. However, the underlying molecular mechanisms of this activation subsequent retinal regeneration remains unclear.

To address this, we performed single-cell RNA sequencing (scRNA-seq) and report remarkable heterogeneity in gene expression within quiescent Müller glia across distinct dorsal, central and ventral retina pools of such cells. Next, we utilized a genetically driven, chemically inducible nitroreductase approach to study Müller glia activation following selective ablation of three distinct photoreceptor subtypes: long wavelength sensitive cones, short wavelength sensitive cones, and rods. There, our data revealed that a region-specific bias in activation of Müller glia exists in the zebrafish retina, and this is independent of the distribution of the ablated cell type across retinal regions. Notably, gene ontology analysis revealed that injury-responsive dorsal and central Müller glia express genes related to dorsal/ventral pattern formation, growth factor activity, and regulation of developmental process. Through scRNA-seq analysis, we identify a shared genetic program underlying initial Müller glia activation and cell cycle entry, followed by differences that drive the fate of regenerating neurons. We observed an initial expression of AP-1 and injury-responsive transcription factors, followed by genes involved in Notch signaling, ribosome biogenesis and gliogenesis, and finally expression of cell cycle, chromatin remodeling and microtubule-associated genes.

Taken together, our findings document the regional specificity of gene expression within quiescent Müller glia and demonstrate unique Müller glia activation and regeneration features following neural ablation. These findings will improve our understanding of the molecular pathways relevant to neural regeneration in the retina.

Chromatin access and epigenetic control over gene expression play important roles in regulating developmental processes. However, little is known about how chromatin access and epigenetic gene silencing influence mature glial cells and retinal regeneration. Herein, we investigate the expression and functions of S-adenosylhomocysteine hydrolase (SAHH; AHCY) and histone methyltransferases (HMTs) during the formation of Müller glia (MG)-derived progenitor cells (MGPCs) in the chick and mouse retinas. In chick, AHCY, AHCYL1 and AHCYL2, and many different HMTs are dynamically expressed by MG and MGPCs in damaged retinas. Inhibition of SAHH reduced levels of H3K27me3 and potently blocks the formation of proliferating MGPCs. By using a combination of single cell RNA-seq and single cell ATAC-seq, we find significant changes in gene expression and chromatin access in MG with SAHH inhibition and NMDA-treatment; many of these genes are associated with glial and neuronal differentiation. A strong correlation across gene expression, chromatin access, and transcription factor motif access in MG was observed for transcription factors known to convey glial identity and promote retinal development. By comparison, in the mouse retina, inhibition of SAHH has no influence on the differentiation of neuron-like cells from Ascl1-overexpressing MG. We conclude that in the chick the activity of SAHH and HMTs are required for the reprogramming of MG into MGPCs by regulating chromatin access to transcription factors associated with glial differentiation and retinal development.

Retinal degenerative diseases, characterized by retinal neuronal death and severe vision loss, affect millions of people worldwide. One of the most promising treatment methods for retinal degenerative diseases is to reprogram non-neuronal cells into stem or progenitor cells, which then have the potential to re-differentiate to replace the dead neurons, thereby promoting retinal regeneration. Müller glia are the major glial cell type and play an important regulatory role in retinal metabolism and retinal cell regeneration. Müller glia can serve as a source of neurogenic progenitor cells in organisms with the ability to regenerate the nervous system. Current evidence points toward the reprogramming process of Müller glia, involving changes in the expression of pluripotent factors and other key signaling molecules that may be regulated by epigenetic mechanisms. This review summarizes recent knowledge of epigenetic modifications involved in the reprogramming process of Müller glia and the subsequent changes to gene expression and the outcomes. In living organisms, epigenetic mechanisms mainly include DNA methylation, histone modification, and microRNA-mediated miRNA degradation, all of which play a crucial role in the reprogramming process of Müller glia. The information presented in this review will improve the understanding of the mechanisms underlying the Müller glial reprogramming process and provide a research basis for the development of Müller glial reprogramming therapy for retinal degenerative diseases.

Recent studies have demonstrated the complex coordination of pro-inflammatory signaling and reactive microglia/macrophage on the formation Müller glial-derived progenitor cells (MGPCs) in the retinas of fish, birds and mice. We generated scRNA-seq libraries to identify transcriptional changes in Müller glia (MG) that result from the depletion of microglia from the chick retina. We found significant changes in different networks of genes in MG in normal and damaged retinas when the microglia are ablated. We identified a failure of MG to upregulate Wnt-ligands, Heparin binding epidermal growth factor (HBEGF), Fibroblast growth factor (FGF), retinoic acid receptors and genes related to Notch-signaling. Inhibition of GSK3β, to simulate Wnt-signaling, failed to rescue the deficit in formation of proliferating MGPCs in damaged retinas missing microglia. By comparison, application of HBEGF or FGF2 completely rescued the formation of proliferating MGPCs in microglia-depleted retinas. Similarly, injection of a small molecule inhibitor to Smad3 or agonist to retinoic acid receptors partially rescued the formation of proliferating MGPCs in microglia-depleted damaged retinas. According to scRNA-seq libraries, patterns of expression of ligands, receptors, signal transducers and/or processing enzymes to cell-signaling via HBEGF, FGF, retinoic acid and TGFβ are rapidly and transiently upregulated by MG after neuronal damage, consistent with important roles for these cell-signaling pathways in regulating the formation of MGPCs. We conclude that quiescent and activated microglia have a significant impact upon the transcriptomic profile of MG. We conclude that signals produced by reactive microglia in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGFβ/Smad3 to promote the reprogramming on MG into proliferating MGPCs.

Nervous system diseases present significant challenges to the neuroscience community due to ethical and practical constraints that limit access to appropriate research materials. Somatic cell reprogramming has been proposed as a novel way to obtain neurons. Various emerging techniques have been used to reprogram mature and differentiated cells into neurons. This review provides an overview of somatic cell reprogramming for neurological research and therapy, focusing on neural reprogramming and generating different neural cell types. We examine the mechanisms involved in reprogramming and the challenges that arise. We herein summarize cell reprogramming strategies to generate neurons, including transcription factors, small molecules, and microRNAs, with a focus on different types of cells.. While reprogramming somatic cells into neurons holds the potential for understanding neurological diseases and developing therapeutic applications, its limitations and risks must be carefully considered. Here, we highlight the potential benefits of somatic cell reprogramming for neurological disease research and therapy. This review contributes to the field by providing a comprehensive overview of the various techniques used to generate neurons by cellular reprogramming and discussing their potential applications.

Müller glia are non-neuronal support cells that play a vital role in the homeostasis of the eye. Their radial-oriented processes span the width of the retina and respond to injury through a cellular response that can be detrimental or protective depending on the context. In some species, protective responses include the expression of stem cell-like genes which help to fuel new neuron formation and even restoration of vision. In many lower vertebrates including fish and amphibians, this response is well documented, however, in mammals it is severely limited. The remarkable plasticity of cellular reprogramming in lower vertebrates has inspired studies in mammals for repairing the retina and restoring sight, and recent studies suggest that mammals are also capable of regeneration, albeit to a lesser degree. Endogenous regeneration, whereby new retinal neurons are created from existing support cells, offers an exciting alternative approach to existing tissue transplant, gene therapy, and neural prosthetic approaches being explored in parallel. This review will highlight the role of Müller glia during retinal injury and repair. In the end, prospects for advancing retinal regeneration research will be considered.

Endogenous regeneration strategies to replace lost neurons hold great promise for treating neurodegenerative disorders. In the majority of cases, neural regeneration is induced by converting resident glial cells into neurogenic precursors. This review will outline how proneural bHLH transcription factors can be used to reprogram glia in the brain and retina into a source for new neurons.

Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in nonmammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. Here, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in Müller glia, including Ascl1, Pou4f2, Islet1, and Atoh1. Using immunohistochemistry, single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing, and electrophysiology, we find that retinal ganglion-like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental retinal ganglion cell TFs.

Unlike mammals, vertebrates such as fishes and frogs exhibit remarkable tissue regeneration including the central nervous system. Retina being part of the central nervous system has attracted the interest of several research groups to explore its regenerative ability in different vertebrate models including mice. Fishes and frogs completely restore the size, shape and tissue structure of an injured retina. Several studies have unraveled molecular mechanisms underlying retina regeneration. In teleosts, soon after injury, the Müller glial cells of the retina reprogram to form a proliferating population of Müller glia-derived progenitor cells capable of differentiating into various neural cell types and Müller glia. In amphibians, the transdifferentiation of retinal pigment epithelium and differentiation of ciliary marginal zone cells contribute to retina regeneration. In chicks and mice, supplementation with external growth factors or genetic modifications cause a partial regenerative response in the damaged retina. The initiation of retina regeneration is achieved through sequential orchestration of gene expression through controlled modulations in the genetic and epigenetic landscape of the progenitor cells. Several developmental biology pathways are turned on during the Müller glia reprogramming, retinal pigment epithelium transdifferentiation and ciliary marginal zone differentiation. Further, several tumorigenic pathways and gene expression events also contribute to the complete regeneration cascade of events. In this review, we address the various retinal injury paradigms and subsequent gene expression events governed in different vertebrate species. Further, we compared how vertebrates such as teleost fishes and amphibians can achieve excellent regenerative responses in the retina compared with their mammalian counterparts.

The regenerative capacity of the vertebrate retina varies substantially across species. Whereas fish and amphibians can regenerate functional retina, mammals do not. In this perspective piece, we outline the various strategies nonmammalian vertebrates use to achieve functional regeneration of vision. We review key differences underlying the regenerative potential across species including the cellular source of postnatal progenitors, the diversity of cell fates regenerated, and the level of functional vision that can be achieved. Finally, we provide an outlook on the field of engineering the mammalian retina to replace neurons lost to injury or disease.

Progressive and irreversible vision loss in mature and aging adults creates a health and economic burden, worldwide. Despite the advancements of many contemporary therapies to restore vision, few approaches have considered the innate benefits of gliosis, the endogenous processes of retinal repair that precede vision loss. Retinal gliosis is fundamentally driven by Müller glia (MG) and is characterized by three primary cellular mechanisms: hypertrophy, proliferation, and migration. In early stages of gliosis, these processes have neuroprotective potential to halt the progression of disease and encourage synaptic activity among neurons. Later stages, however, can lead to glial scarring, which is a hallmark of disease progression and blindness. As a result, the neuroprotective abilities of MG have remained incompletely explored and poorly integrated into current treatment regimens. Bioengineering studies of the intrinsic behaviors of MG hold promise to exploit glial reparative ability, while repressing neuro-disruptive MG responses. In particular, recent in vitro systems have become primary models to analyze individual gliotic processes and provide a stepping stone for in vivo strategies. This review highlights recent studies of MG gliosis seeking to harness MG neuroprotective ability for regeneration using contemporary biotechnologies. We emphasize the importance of studying gliosis as a reparative mechanism, rather than disregarding it as an unfortunate clinical prognosis in diseased retina.

In mammals, photoreceptor loss causes permanent blindness, but in zebrafish (Danio rerio), photoreceptor loss reprograms Müller glia to function as stem cells, producing progenitors that regenerate photoreceptors. MicroRNAs (miRNAs) regulate CNS neurogenesis, but the roles of miRNAs in injury-induced neuronal regeneration are largely unknown. In the embryonic zebrafish retina, miR-18a regulates photoreceptor differentiation. The purpose of the current study was to determine, in zebrafish, the function of miR-18a during injury-induced photoreceptor regeneration. RT-qPCR, in situ hybridization, and immunohistochemistry showed that miR-18a expression increases throughout the retina between 1 and 5 days post-injury (dpi). To test miR-18a function during photoreceptor regeneration, we used homozygous miR-18a mutants (miR-18ami5012), and knocked down miR-18a with morpholino oligonucleotides. During photoreceptor regeneration, miR-18ami5012 retinas have fewer mature photoreceptors than WT at 7 and 10 dpi, but there is no difference at 14 dpi, indicating that photoreceptor regeneration is delayed. Labeling dividing cells with 5-bromo-2'-deoxyuridine (BrdU) showed that at 7 and 10 dpi, there are excess dividing progenitors in both mutants and morphants, indicating that miR-18a negatively regulates injury-induced proliferation. Tracing 5-ethynyl-2'-deoxyuridine (EdU) and BrdU-labeled cells showed that in miR-18ami5012 retinas excess progenitors migrate to other retinal layers in addition to the photoreceptor layer. Inflammation is critical for photoreceptor regeneration, and RT-qPCR showed that in miR-18ami5012 retinas, inflammatory gene expression and microglia activation are prolonged. Suppressing inflammation with dexamethasone rescues the miR-18ami5012 phenotype. Together, these data show that in the injured zebrafish retina, disruption of miR-18a alters proliferation, inflammation, the microglia/macrophage response, and the timing of photoreceptor regeneration.