Regeneration of Inner Retinal Neurons after Intravitreal Injection of Ouabain in Zebrafish

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279

Key to successful retina regeneration in zebrafish are Müller glia (MG) that respond to retinal injury by dedifferentiating into a cycling population of retinal progenitors. Although recent studies have identified several genes involved in retina regeneration, the signaling mechanisms underlying injury-dependent MG proliferation have remained elusive. Here we report that canonical Wnt signaling controls the proliferation of MG-derived retinal progenitors. We found that injury-dependent induction of Ascl1a suppressed expression of the Wnt signaling inhibitor, Dkk, and induced expression of the Wnt ligand, Wnt4a. Genetic and pharmacological inhibition of Wnt signaling suppressed injury-dependent proliferation of MG-derived progenitors. Remarkably, in the uninjured retina, glycogen synthase kinase-3β (GSK-3β) inhibition was sufficient to stimulate MG dedifferentiation and the formation of multipotent retinal progenitors that were capable of differentiating into all major retinal cell types. Importantly, Ascl1a expression was found to contribute to the multipotential character of these progenitors. Our data suggest that Wnt signaling and GSK-3β inhibition, in particular, are crucial for successful retina regeneration.pyrvinium | XAV939 | transgenic zebrafish | heat shock | frizzled

mentioning

confidence: 99%

Ascl1a/Dkk/β-catenin signaling pathway is necessary and glycogen synthase kinase-3β inhibition is sufficient for zebrafish retina regeneration

Ramachandran

Zhao

Goldman

2011

195

279

“…Unlike mammals, zebrafish can regenerate multiple tissues, including the retina. During zebrafish retina regeneration, Müller glia (MG) reprogram to generate progenitor cells (MGPCs) capable of replacing all lost neural cell types (9)(10)(11)(12). The role that MG play during this regenerative event can be roughly divided into three phases: the replication-independent transition of MG to a MPGC occurring by 2 d postinjury (dpi), the assymetric division and proliferative amplification of MGPCs (13) between 2 and 7 dpi, and the differentiation of these progenitors following 7 dpi (Fig.…”

mentioning

confidence: 99%

Analysis of DNA methylation reveals a partial reprogramming of the Müller glia genome during retina regeneration

Powell

Grant

Cornblath

et al. 2013

110

138

Upon retinal injury, zebrafish Müller glia (MG) transition from a quiescent supportive cell to a progenitor cell (MGPC). This event is accompanied by the induction of key transcription and pluripotency factors. Because somatic cell reprogramming during induced pluripotent stem cell generation is accompanied by changes in DNA methylation, especially in pluripotency factor gene promoters, we were interested in determining whether DNA methylation changes also underlie MG reprogramming following retinal injury. Consistent with this idea, we found that genes encoding components of the DNA methylation/demethylation machinery were induced in MGPCs and that manipulating MGPC DNA methylation with 5-aza-2′-deoxycytidine altered their properties. A comprehensive analysis of the DNA methylation landscape as MG reprogram to MGPCs revealed that demethylation predominates at early times, whereas levels of de novo methylation increase at later times. We found that these changes in DNA methylation were largely independent of Apobec2 protein expression. A correlation between promoter DNA demethylation and injurydependent gene induction was noted. In contrast to induced pluripotent stem cell formation, we found that pluripotency factor gene promoters were already hypomethylated in quiescent MG and remained unchanged in MGPCs. Interestingly, these pluripotency factor promoters were also found to be hypomethylated in mouse MG. Our data identify a dynamic DNA methylation landscape as zebrafish MG transition to an MGPC and suggest that DNA methylation changes will complement other regulatory mechanisms to ensure gene expression programs controlling MG reprogramming are appropriately activated during retina regeneration.repair | multipotent | Ascl1 | bisulfite sequencing | DNA sequencing I nduced pluripotent stem cells (iPSCs) can be generated through the forced expression of pluripotency factor genes, such as Oct4, Klf4, Sox2, c-Myc, Lin28, and Nanog, which are normally expressed in embryonic stem cells (ESCs) (1, 2). Pluripotency factor gene expression in ESCs and iPSCs is associated with chromatin that is in an "open" accessible state, whereas their repression in somatic cells is associated with less accessible, condensed chromatin (3). DNA methylation has a significant impact on chromatin structure (3). DNA demethylation of pluripotency factor promoter regions is correlated with increased expression during iPSC formation (4, 5). Similar epigenetic changes are seen in other cellular reprogramming events such as nuclear transfer (6), heterokaryon formation (7), and carcinogenesis (8).Tissue regeneration provides another avenue to study cellular reprogramming. Unlike mammals, zebrafish can regenerate multiple tissues, including the retina. During zebrafish retina regeneration, Müller glia (MG) reprogram to generate progenitor cells (MGPCs) capable of replacing all lost neural cell types (9-12). The role that MG play during this regenerative event can be roughly divided into three phases: the replication-independent transition of MG to...

“…The neural stem cells in the retina arise from differentiated Müller glia, which respond to local retinal injuries by dedifferentiation, proliferation, and production of multipotent neuronal progenitors (retinal stem cells) that can regenerate all types of retinal neurons (17)(18)(19). To discover genes expressed in injury-activated neurogenic Müller glial cells that activate stem cell properties and trigger a neurogenic program, we generated transcriptional profiles of isolated fluorescent-tagged Müller glial cells from light-lesioned adult transgenic zebrafish retinas during the early stages of photoreceptor regeneration.…”

mentioning

confidence: 99%

Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish

Qin

Barthel

Raymond

2009

131

171

In a microarray-based gene profiling analysis of Mü ller glia-derived retinal stem cells in light-damaged retinas from adult zebrafish, we found that 2 genes required for regeneration of fin and heart tissues in zebrafish, hspd1 (heat shock 60-kDa protein 1) and mps1 (monopolar spindle 1), were up-regulated. Expression of both genes in the neurogenic Mü ller glia and progenitors was independently verified by quantitative reverse transcriptase PCR and in situ hybridization. Functional analysis of temperature-sensitive mutants of hspd1 and mps1 revealed that both are necessary for Mü ller glia-based cone photoreceptor regeneration in adult zebrafish retina. In the amputated fin, hspd1 is required for the induction of mesenchymal stem cells and blastema formation, whereas mps1 is required at a later step for rapid cell proliferation and outgrowth. This temporal sequence of hspd1 and mps1 function is conserved in the regenerating retina. Comparison of gene expression profiles from regenerating zebrafish retina, caudal fin, and heart muscle revealed additional candidate genes potentially implicated in injury-induced epimorphic regeneration in diverse zebrafish tissues.T he study of regeneration has long fascinated biologists and has lately experienced a renaissance associated with growing interest in regenerative medicine and the therapeutic potential of stem cells. Zebrafish (Danio rerio) are an ideal genetic model for studying regeneration in vertebrates (1) because they have remarkable capabilities to regenerate fins (2), heart muscle (3), and nervous tissues (4-7) following injury. A forward mutagenesis screen for temperature-sensitive mutations that interfere with regeneration of amputated caudal fin identified several genes whose functions are critical for specific steps in fin regeneration, including mps1 (monopolar spindle 1, also called ttk, a kinase required for mitotic checkpoint regulation), hspd1 (heat shock 60-kDa protein 1, a mitochondrial chaperone), and fgf20 (fibroblast growth factor 20) (8-10). In addition, gene profiling analysis of regenerating tissues has provided lists of candidate genes associated with regeneration in fin (11), heart (12), and neural retina (13-15).The regeneration of retinal neurons in adult zebrafish is an especially powerful model for studying regeneration of neuronal tissues; laminar retinal architecture and visual function are restored following damage inflicted by surgical lesions, neurotoxins, and laser or photic lesions of retina (16). The neural stem cells in the retina arise from differentiated Müller glia, which respond to local retinal injuries by dedifferentiation, proliferation, and production of multipotent neuronal progenitors (retinal stem cells) that can regenerate all types of retinal neurons (17-19). To discover genes expressed in injury-activated neurogenic Müller glial cells that activate stem cell properties and trigger a neurogenic program, we generated transcriptional profiles of isolated fluorescent-tagged Müller glial cells from light-lesioned adult ...