Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes through Müller glia (MG) reprogramming and asymmetric cell division that produces a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, do MG reprogram to a developmental retinal progenitor cell (RPC) state? Second, to what extent does regeneration recapitulate retinal development? And finally, does loss of different retinal cell subtypes induce unique MG regeneration responses? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. Here we show that injury induces MG to reprogram to a state similar to late-stage RPCs. However, there are major transcriptional differences between MGPCs and RPCs, as well as major transcriptional differences between activated MG and MGPCs when different retinal cell subtypes are damaged. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes.

Her6 and Prox1a are novel regulators of photoreceptor regeneration in the zebrafish retina.

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.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Common and divergent gene regulatory networks control injury-induced and developmental neurogenesis in zebrafish retina.

Following acute retinal damage, zebrafish possess the ability to regenerate all neuronal subtypes. This regeneration requires Müller glia (MG) to reprogram and divide asymmetrically to produce a multipotent Müller glia-derived neuronal progenitor cell (MGPC). This raises three key questions. First, does loss of different retinal cell subtypes induce unique MG regeneration responses? Second, do MG reprogram to a developmental retinal progenitor cell state? And finally, to what extent does regeneration recapitulate retinal development? We examined these questions by performing single-nuclear and single-cell RNA-Seq and ATAC-Seq in both developing and regenerating retinas. While MG reprogram to a state similar to late-stage retinal progenitors in developing retinas, there are transcriptional differences between reprogrammed MG/MGPCs and late progenitors, as well as reprogrammed MG in outer and inner retinal damage models. Validation of candidate genes confirmed that loss of different subtypes induces differences in transcription factor gene expression and regeneration outcomes. This work identifies major differences between gene regulatory networks activated following the selective loss of different subtypes of retina neurons, as well as between retinal regeneration and development.

Extracellular Matrix: The Unexplored Aspects of Retinal Pathologies and Regeneration.

Nearly a billion people worldwide are affected by vision-impairing conditions, with retinal degenerative diseases being a major cause of blindness. Unfortunately, such diseases are often permanent and progressive, resulting in further degeneration and loss of sight, due to the human retina possessing little, if any, regenerative capacity. Despite numerous efforts and great progress being made to understand the molecular mechanisms of these diseases and possible therapies, the majority of investigations focused on cell-intrinsic factors. However, the microenvironment surrounding retinal cells throughout these processes also plays an important role, though our current understanding of its involvement remains limited. Here we present a brief overview of the current state of the field of extracellular matrix studies within the retina and its potential roles in retinal diseases and potential therapeutic approaches.

Live Cell Imaging of Dynamic Processes in Adult Zebrafish Retinal Cross-Section Cultures.

Following retinal injury, zebrafish possess the remarkable capacity to endogenously regenerate lost retinal neurons from Müller glia-derived neuronal progenitor cells. Additionally, neuronal cell types that are undamaged and persist in the injured retina are also produced. Thus, the zebrafish retina is an excellent system to study the integration of all neuronal cell types into an existing neuronal circuit. The few studies that examined axonal/dendritic outgrowth and the establishment of synaptic contacts by regenerated neurons predominantly utilized fixed tissue samples. We recently established a flatmount culture model to monitor Müller glia nuclear migration in real time by two-photon microscopy. However, in retinal flatmounts, z-stacks of the entire retinal z-dimension have to be acquired to image cells that extend through parts or the entirety of the neural retina, such as bipolar cells and Müller glia, respectively. Cellular processes with fast kinetics might thus be missed. Therefore, we generated a retinal cross-section culture from light-damaged zebrafish to image the entire Müller glia in one z-plane. Isolated dorsal retinal hemispheres were cut into two dorsal quarters and mounted with the cross-section view facing the coverslips of culture dishes, which allowed monitoring Müller glia nuclear migration using confocal microscopy. Confocal imaging of cross-section cultures is ultimately also applicable to live cell imaging of axon/dendrite formation of regenerated bipolar cells, while the flatmount culture model will be more suitable to monitor axon outgrowth of ganglion cells.

Clcf1/Crlf1a-mediated signaling is neuroprotective and required for Müller glia proliferation in the light-damaged zebrafish retina.

Zebrafish possess the innate ability to fully regenerate any neurons lost following a retinal injury. This response is mediated by Müller glia that reprogram and divide asymmetrically to produce neuronal precursor cells that differentiate into the lost neurons. However, little is understood about the early signals that induce this response. Ciliary neurotrophic factor (CNTF) was previously shown to be both neuroprotective and pro-proliferative within the zebrafish retina, however CNTF is not expressed following injury. Here we demonstrate that alternative ligands of the Ciliary neurotrophic factor receptor (CNTFR), such as Cardiotrophin-like cytokine factor 1 (Clcf1) and Cytokine receptor-like factor 1a (Crlf1a), are expressed within Müller glia of the light-damaged retina. We found that CNTFR, Clcf1, and Crlf1a are required for Müller glia proliferation in the light-damaged retina. Furthermore, intravitreal injection of CLCF1/CRLF1 protected against rod photoreceptor cell death in the light-damaged retina and induced proliferation of rod precursor cells in the undamaged retina, but not Müller glia. While rod precursor cell proliferation was previously shown to be Insulin-like growth factor 1 receptor (IGF-1R)-dependent, co-injection of IGF-1 with CLCF1/CRLF1 failed to induce further proliferation of either Müller glia or rod precursor cells. Together, these findings demonstrate that CNTFR ligands have a neuroprotective effect and are required for induction of Müller glia proliferation in the light-damaged zebrafish retina.

Different inflammation responses modulate Müller glia proliferation in the acute or chronically damaged zebrafish retina.

Unlike mammals, zebrafish regenerate in response to retinal damage. Because microglia are activated by retinal damage, we investigated their role during regeneration following either acute or chronic damage. At three weeks post-fertilization (wpf), both wild-type fish exhibiting NMDA-induced acute ganglion and amacrine cell death and gold rush (gosh) mutant fish possessing chronic cone photoreceptor degeneration displayed reactive microglia/macrophages and Müller glia proliferation. Dexamethasone-treated retinas, to inhibit the immune response, lacked reactive microglia/macrophages and possessed fewer PCNA-positive cells, while LPS treatment increased microglia/macrophages and PCNA-labeled cells. NMDA-injured retinas upregulated expression of il-1ß and tnfα pro-inflammatory cytokine genes, followed by increased expression of il-10 and arg1 anti-inflammatory/remodeling cytokine genes. A transient early TNFα pro-inflammatory microglia/macrophage population was visualized in NMDA-damaged retinas. In contrast, gosh mutant retinas exhibited a slight increase of pro-inflammatory cytokine gene expression concurrently with a greater increased anti-inflammatory/remodeling cytokine gene expression. Few TNFα pro-inflammatory microglia/macrophages were observed in the gosh retina. Understanding why acute and chronic damage results in different inflammation profiles and their effects on regulating zebrafish retinal regeneration would provide important clues toward improving therapeutic strategies for repairing injured mammalian tissues.

Notch Inhibition Promotes Regeneration and Immunosuppression Supports Cone Survival in a Zebrafish Model of Inherited Retinal Dystrophy.

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as retinitis pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Müller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, in zebrafish models of retinal degeneration, photoreceptor death does not induce the MG to reprogram and regenerate lost cells. Here, we used male and female zebrafish cep290 mutants to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to prelesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.SIGNIFICANCE STATEMENT Inherited retinal degenerations (IRDs) are genetic diseases that lead to the progressive loss of photoreceptors and the permanent loss of vision. Zebrafish can regenerate photoreceptors after acute injury by reprogramming Müller glia (MG) into stem-like cells that produce retinal progenitors, but this regenerative process fails to occur in zebrafish models of IRDs. Here, we show that Notch pathway inhibition can promote photoreceptor regeneration in models of progressive degeneration and that immunosuppression can prevent photoreceptor loss. These results offer insight into the pathways that promote MG-dependent regeneration and the role of inflammation in photoreceptor degeneration.

Iron contributes to photoreceptor degeneration and Müller glia proliferation in the zebrafish light-treated retina.

Zebrafish possess the ability to completely regenerate the retina following injury, however little is understood about the damage signals that contribute to inducing Müller glia reprogramming and proliferation to regenerate lost neurons. Multiple studies demonstrated that iron contributes to various retinal injuries, however no link has been shown between iron and zebrafish retinal regeneration. Here we demonstrate that Müller glia exhibit transcriptional changes following injury to regulate iron levels within the retina, allowing for increased iron uptake and decreased export. The response of the zebrafish retina to intravitreal iron injection was then characterized, showing that ferrous, and not ferric, iron induces retinal cell death. Additionally, iron chelation resulted in decreased numbers of TUNEL-positive photoreceptors and fewer proliferating Müller glia. Despite the contribution of iron to retinal cell death, inhibition of ferroptosis did not significantly reduce cell death following light treatment. Finally, we demonstrate that both the anti-ferroptotic protein Glutathione peroxidase 4b and the Transferrin receptor 1b are required for Müller glia proliferation following light damage. Together these findings show that iron contributes to cell death in the light-damaged retina and is essential for inducing the Müller glia regeneration response.

Retinal regeneration requires dynamic Notch signaling.

Retinal damage in the adult zebrafish induces Müller glia reprogramming to produce neuronal progenitor cells that proliferate and differentiate into retinal neurons. Notch signaling, which is a fundamental mechanism known to drive cell-cell communication, is required to maintain Müller glia in a quiescent state in the undamaged retina, and repression of Notch signaling is necessary for Müller glia to reenter the cell cycle. The dynamic regulation of Notch signaling following retinal damage also directs proliferation and neurogenesis of the Müller glia-derived progenitor cells in a robust regeneration response. In contrast, mammalian Müller glia respond to retinal damage by entering a prolonged gliotic state that leads to additional neuronal death and permanent vision loss. Understanding the dynamic regulation of Notch signaling in the zebrafish retina may aid efforts to stimulate Müller glia reprogramming for regeneration of the diseased human retina. Recent findings identified DeltaB and Notch3 as the ligand-receptor pair that serves as the principal regulators of zebrafish Müller glia quiescence. In addition, multi-omics datasets and functional studies indicate that additional Notch receptors, ligands, and target genes regulate cell proliferation and neurogenesis during the regeneration time course. Still, our understanding of Notch signaling during retinal regeneration is limited. To fully appreciate the complex regulation of Notch signaling that is required for successful retinal regeneration, investigation of additional aspects of the pathway, such as post-translational modification of the receptors, ligand endocytosis, and interactions with other fundamental pathways is needed. Here we review various modes of Notch signaling regulation in the context of the vertebrate retina to put recent research in perspective and to identify open areas of inquiry.

Sex Differences in Behavioral Symptoms and the Levels of Circulating GFAP, Tau, and NfL in Patients With Traumatic Brain Injury.

Traumatic brain injury (TBI) affects millions of Americans each year and has been shown to disproportionately impact those subject to greater disparities in health. Female sex is one factor that has been associated with disparities in health outcomes, including in TBI, but sex differences in biomarker levels and behavioral outcomes after TBI are underexplored. This study included participants with both blunt and blast TBI with majority rating their TBI as mild. Time since injury was 5.4 (2.0, 15.5) years for females and 6.8 (2.4, 11.3) years for males. The aim of this cross sectional study is to investigate the relationship between postconcussive, depression, and post-traumatic stress disorder (PTSD) symptoms, as well as health related quality of life (HRQOL), and the levels of glial fibrillary acidic protein (GFAP), total tau (t-tau), neurofilament light chain (NfL), and ubiquitin C-terminal hydrolase-L1 (UCH-L1). Behavioral outcomes were evaluated with the Neurobehavioral Symptom Inventory (NSI), Patient Health Questionnaire-9 (PHQ-9), PTSD Checklist- Civilian Version (PCL-C), short form (SF)-36, and plasma levels of total tau, GFAP, NfL, and UCHL-1 measured with the Simoa-HDX. We observed that females had significantly higher levels of GFAP and tau (ps < 0.05), and higher PHQ-9 scores, NSI total scores, NSI- vestibular, NSI-somatosensory, NSI-affective sub-scale scores (ps < 0.05)), than males. In addition, females had lower scores in HRQOL outcomes of role limitations due to emotional problems, vitality, emotional well-being, social functioning, and pain compared to males (ps < 0.05). Correlation analysis showed positive associations between levels of tau and the NSI-total and NSI-cognitive sub-scale scores (ps < 0.05) in females. No significant associations were found for NfL or GFAP with NSI scores. For female participants, negative correlations were observed between tau and NfL concentrations and the SF-36 physical function subscale (ps < 0.05), as well as tau and the social function subscale (p < 0.001), while GFAP levels positively correlated with role limitations due to emotional problems (p = 0.004). No significant associations were observed in males. Our findings suggest that sex differences exist in TBI-related behavioral outcomes, as well as levels of biomarkers associated with brain injury, and that the relationship between biomarker levels and behavioral outcomes is more evident in females than males. Future studies are warranted to corroborate these results, and to determine the implications for prognosis and treatment. The identification of candidate TBI biomarkers may lead to development of individualized treatment guidelines.

Zebrafish Blunt-Force TBI Induces Heterogenous Injury Pathologies That Mimic Human TBI and Responds with Sonic Hedgehog-Dependent Cell Proliferation across the Neuroaxis.

Blunt-force traumatic brain injury (TBI) affects an increasing number of people worldwide as the range of injury severity and heterogeneity of injury pathologies have been recognized. Most current damage models utilize non-regenerative organisms, less common TBI mechanisms (penetrating, chemical, blast), and are limited in scalability of injury severity. We describe a scalable blunt-force TBI model that exhibits a wide range of human clinical pathologies and allows for the study of both injury pathology/progression and mechanisms of regenerative recovery. We modified the Marmarou weight drop model for adult zebrafish, which delivers a scalable injury spanning mild, moderate, and severe phenotypes. Following injury, zebrafish display a wide range of severity-dependent, injury-induced pathologies, including seizures, blood-brain barrier disruption, neuroinflammation, edema, vascular injury, decreased recovery rate, neuronal cell death, sensorimotor difficulties, and cognitive deficits. Injury-induced pathologies rapidly dissipate 4-7 days post-injury as robust cell proliferation is observed across the neuroaxis. In the cerebellum, proliferating nestin:GFP-positive cells originated from the cerebellar crest by 60 h post-injury, which then infiltrated into the granule cell layer and differentiated into neurons. Shh pathway genes increased in expression shortly following injury. Injection of the Shh agonist purmorphamine in undamaged fish induced a significant proliferative response, while the proliferative response was inhibited in injured fish treated with cyclopamine, a Shh antagonist. Collectively, these data demonstrate that a scalable blunt-force TBI to adult zebrafish results in many pathologies similar to human TBI, followed by recovery, and neuronal regeneration in a Shh-dependent manner.

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

A Scalable Model to Study the Effects of Blunt-Force Injury in Adult Zebrafish.

Blunt-force traumatic brain injuries (TBI) are the most common form of head trauma, which spans a range of severities and results in complex and heterogenous secondary effects. While there is no mechanism to replace or regenerate the lost neurons following a TBI in humans, zebrafish possess the ability to regenerate neurons throughout their body, including the brain. To examine the breadth of pathologies exhibited in zebrafish following a blunt-force TBI and to study the mechanisms underlying the subsequent neuronal regenerative response, we modified the commonly used rodent Marmarou weight drop for the use in adult zebrafish. Our simple blunt-force TBI model is scalable, inducing a mild, moderate, or severe TBI, and recapitulates many of the phenotypes observed following human TBI, such as contact- and post-traumatic seizures, edema, subdural and intracerebral hematomas, and cognitive impairments, each displayed in an injury severity-dependent manner. TBI sequelae, which begin to appear within minutes of the injury, subside and return to near undamaged control levels within 7 days post-injury. The regenerative process begins as early as 48 hours post-injury (hpi), with the peak cell proliferation observed by 60 hpi. Thus, our zebrafish blunt-force TBI model produces characteristic primary and secondary injury TBI pathologies similar to human TBI, which allows for investigating disease onset and progression, along with the mechanisms of neuronal regeneration that is unique to zebrafish.

Prophylactic Activation of Shh Signaling Attenuates TBI-Induced Seizures in Zebrafish by Modulating Glutamate Excitotoxicity through Eaat2a.

Approximately 2 million individuals experience a traumatic brain injury (TBI) every year in the United States. Secondary injury begins within minutes after TBI, with alterations in cellular function and chemical signaling that contribute to excitotoxicity. Post-traumatic seizures (PTS) are experienced in an increasing number of TBI individuals that also display resistance to traditional anti-seizure medications (ASMs). Sonic hedgehog (Shh) is a signaling pathway that is upregulated following central nervous system damage in zebrafish and aids injury-induced regeneration. Using a modified Marmarou weight drop on adult zebrafish, we examined PTS following TBI and Shh modulation. We found that inhibiting Shh signaling by cyclopamine significantly increased PTS in TBI fish, prolonged the timeframe PTS was observed, and decreased survival across all TBI severities. Shh-inhibited TBI fish failed to respond to traditional ASMs, but were attenuated when treated with CNQX, which blocks ionotropic glutamate receptors. We found that the Smoothened agonist, purmorphamine, increased Eaat2a expression in undamaged brains compared to untreated controls, and purmorphamine treatment reduced glutamate excitotoxicity following TBI. Similarly, purmorphamine reduced PTS, edema, and cognitive deficits in TBI fish, while these pathologies were increased and/or prolonged in cyclopamine-treated TBI fish. However, the increased severity of TBI phenotypes with cyclopamine was reduced by cotreating fish with ceftriaxone, which induces Eaat2a expression. Collectively, these data suggest that Shh signaling induces Eaat2a expression and plays a role in regulating TBI-induced glutamate excitotoxicity and TBI sequelae.

Notch3 and DeltaB maintain Müller glia quiescence and act as negative regulators of regeneration in the light-damaged zebrafish retina.

Damage to the zebrafish retina stimulates resident Müller glia to reprogram, reenter the cell cycle, divide asymmetrically, and produce neuronal progenitor cells that amplify and differentiate into the lost neurons. The transition from quiescent to proliferative Müller glia involves both positive and negative regulators. We previously demonstrated that the Notch signaling pathway represses retinal regeneration by maintaining Müller glia quiescence in zebrafish. Here we examine which Notch receptor is necessary to maintain quiescence. Quantitative RT-PCR and RNA-Seq analyses reveal that notch3 is expressed in the undamaged retina and is downregulated in response to light damage. Additionally, Notch3 protein is expressed in quiescent Müller glia of the undamaged retina, is downregulated as Müller glia proliferate, and is reestablished in the Müller glia. Knockdown of Notch3 is sufficient to induce Müller glia proliferation in undamaged retinas and enhances proliferation during light damage. Alternatively, knockdown of Notch1a, Notch1b, or Notch2 decreases the number of proliferating cells during light damage, suggesting that Notch signaling is also required for proliferation during retinal regeneration. We also knockdown the zebrafish Delta and Delta-like proteins, ligands for the Notch receptors, and find that the deltaB morphant possesses an increased number of proliferating cells in the light-damaged retina. As with Notch3, knockdown of DeltaB is sufficient to induce Müller glia proliferation in the absence of light damage. Taken together, the negative regulation of Müller glia proliferation in zebrafish retinal regeneration is mediated by Notch3 and DeltaB.

Gene regulatory networks controlling vertebrate retinal regeneration.

Injury induces retinal Müller glia of certain cold-blooded vertebrates, but not those of mammals, to regenerate neurons. To identify gene regulatory networks that reprogram Müller glia into progenitor cells, we profiled changes in gene expression and chromatin accessibility in Müller glia from zebrafish, chick, and mice in response to different stimuli. We identified evolutionarily conserved and species-specific gene networks controlling glial quiescence, reactivity, and neurogenesis. In zebrafish and chick, the transition from quiescence to reactivity is essential for retinal regeneration, whereas in mice, a dedicated network suppresses neurogenic competence and restores quiescence. Disruption of nuclear factor I transcription factors, which maintain and restore quiescence, induces Müller glia to proliferate and generate neurons in adult mice after injury. These findings may aid in designing therapies to restore retinal neurons lost to degenerative diseases.

Reprogramming Müller Glia to Regenerate Retinal Neurons.

In humans, various genetic defects or age-related diseases, such as diabetic retinopathies, glaucoma, and macular degeneration, cause the death of retinal neurons and profound vision loss. One approach to treating these diseases is to utilize stem and progenitor cells to replace neurons in situ, with the expectation that new neurons will create new synaptic circuits or integrate into existing ones. Reprogramming non-neuronal cells in vivo into stem or progenitor cells is one strategy for replacing lost neurons. Zebrafish have become a valuable model for investigating cellular reprogramming and retinal regeneration. This review summarizes our current knowledge regarding spontaneous reprogramming of Müller glia in zebrafish and compares this knowledge to research efforts directed toward reprogramming Müller glia in mammals. Intensive research using these animal models has revealed shared molecular mechanisms that make Müller glia attractive targets for cellular reprogramming and highlighted the potential for curing degenerative retinal diseases from intrinsic cellular sources.

Inflammation and matrix metalloproteinase 9 (Mmp-9) regulate photoreceptor regeneration in adult zebrafish.

Brain injury activates complex inflammatory signals in dying neurons, surviving neurons, and glia. Here, we establish that inflammation regulates the regeneration of photoreceptors in the zebrafish retina and determine the cellular expression and function of the inflammatory protease, matrix metalloproteinase 9 (Mmp-9), during this regenerative neurogenesis. Following photoreceptor ablation, anti-inflammatory treatment suppresses the number of injury-induced progenitors and regenerated photoreceptors. Upon photoreceptor injury, mmp-9 is induced in Müller glia and Müller glia-derived photoreceptor progenitors. Deleting mmp-9 results in over production of injury-induced progenitors and regenerated photoreceptors, but over time the absence of Mmp-9 compromises the survival of the regenerated cones. At all time-points studied, the levels of tnf-α are significantly elevated in mutant retinas. Anti-inflammatory treatment in mutants rescues the defects in cone survival. These data provide a link between injury-induced inflammation in the vertebrate CNS, Mmp-9 function during neuronal regeneration and the requirement of Mmp-9 for the survival of regenerated cones.