RESUMEN
Müller glia can act as endogenous stem cells and regenerate the missing neurons in the injured or degenerating retina in lower vertebrates. However, mammalian Müller glia, although can sometimes express stem cell markers and specific neuronal proteins in response to injury or degeneration, do not differentiate into functional neurons. We asked whether bFGF and insulin would stimulate the Müller glia to migrate, proliferate and differentiate into photoreceptors in rd1 mouse. We administered single or repeated (two or three) intravitreal injections of basic fibroblast growth factor (bFGF;200⯵g) and insulin (2⯵g) in 2-week-old rd1 mice. Müller glia were checked for proliferation, migration and differentiation using immunostaining. A single injection resulted within 5 days in a decrease in the numbers of Müller glia in the inner nuclear layer (INL) and a corresponding increase in the outer nuclear layer (ONL). The total number of Müller glia in the INL and ONL was unaltered, suggesting that they did not proliferate, but migrated from INL to ONL. However, maintaining the Müller cells in the ONL for two weeks or longer required repeated injections of bFGF and insulin. Interestingly, all Müller cells in the ONL expressed chx10, a stem cell marker. We did not find any immunolabeling for rhodopsin, m-opsin or s-opsin in the Müller glia in the ONL.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Células Ependimogliales/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Insulina/administración & dosificación , Neuroglía/efectos de los fármacos , Células Fotorreceptoras/efectos de los fármacos , Animales , Movimiento Celular/fisiología , Células Ependimogliales/química , Células Ependimogliales/metabolismo , Inyecciones Intravítreas , Ratones , Ratones Endogámicos CBA , Ratones Transgénicos , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/fisiología , Neuroglía/química , Neuroglía/metabolismo , Células Fotorreceptoras/química , Células Fotorreceptoras/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patologíaRESUMEN
Tanycytes are highly specialized ependymal cells that line the bottom and the lateral walls of the third ventricle. In contact with the cerebrospinal fluid through their cell bodies, they send processes into the arcuate nucleus, the ventromedial nucleus, and the dorsomedial nucleus of the hypothalamus. In the present work, we combined transgenic and immunohistochemical approaches to investigate the neuroanatomical associations between tanycytes and neural cells present in the hypothalamic parenchyma, in particular in the arcuate nucleus. The specific expression of tdTomato in tanycytes first allowed the observation of peculiar subcellular protrusions along tanycyte processes and at their endfeet such as spines, swelling, en passant boutons, boutons, or claws. Interestingly, these protrusions contact different neural cells in the brain parenchyma including blood vessels and neurons, and in particular NPY and POMC neurons in the arcuate nucleus. Using both fluorescent and electron microscopy, we finally observed that these tanycyte protrusions contain ribosomes, mitochondria, diverse vesicles, and transporters, suggesting dense tanycyte/neuron and tanycyte/blood vessel communications. Altogether, our results lay the neuroanatomical basis for tanycyte/neural cell interactions, which will be useful to further understand cell-to-cell communications involved in the regulation of neuroendocrine functions.
Asunto(s)
Células Ependimogliales/ultraestructura , Hipotálamo/ultraestructura , Neuronas/ultraestructura , Tejido Parenquimatoso/ultraestructura , Animales , Células Ependimogliales/química , Cobayas , Humanos , Hipotálamo/química , Hipotálamo/citología , Masculino , Ratones , Ratones Transgénicos , Neuronas/química , Tejido Parenquimatoso/química , Tejido Parenquimatoso/citología , ConejosRESUMEN
Experimental spectra of Müller cell (MC) intermediate filaments (IFs) isolated from porcine retina are reported in this work. The absorption spectra recorded at different MC IF concentrations were used to estimate their absorption cross-sections at different wavelengths. The average absorption cross-section of a single MC IF was ca. (0.97 2.01) × 10-10 cm2 in the 650-445 nm spectral range. To interpret these experimental absorption spectra, we made ab initio calculations of the optical spectra of α-helix polypeptides, and also used a simplified theoretical approach that modeled an IF by a conductive wire. The energy spectra of the refractive index, extinction coefficient (absorption cross-section), energy loss and reflectivity functions for different photon polarizations, with strong anisotropy with respect to the system axis, were calculated ab initio for polyglycine α-helix molecule containing 1000 glycine residues. Strong anisotropy of these parameters was explained by photons interacting with different electronic transitions. Note that similarly strong anisotropy was also obtained for the optical absorption cross-sections in the simplified model. Both modeling approaches were used for calculating the absorption cross-sections of interest. As a result, the absorption cross-section for photons propagating axially along MC IFs was much larger than their geometrical cross-section. The latter result was explained taking into account the density of electronic states, with numerous electrons contributing to the transition intensity at a given energy. We found that the simple conductive wire model describes the MC IF absorption spectrum better than the ab initio spectra. The latter conclusion was explained by the limitations of ab initio analysis, which only took into account one α-helix with 1000 aminoacids, whereas each porcine Müller cell IF is assembled of thousands of protein molecules, reaching the total length of ca. 100 µm. The presently reported results contribute to the understanding of the quantum mechanism of high-contrast vision of vertebrate eyes.
Asunto(s)
Células Ependimogliales/química , Filamentos Intermedios/química , Animales , Células Cultivadas , Péptidos/química , Conformación Proteica en Hélice alfa , Retina/citología , Espectrofotometría , PorcinosRESUMEN
Tanycytes are radial glial cells located in the mediobasal hypothalamus. Recent studies have proposed that tanycytes play an important role in hypothalamic control of energy homeostasis, although this has not been directly tested. Here, we report the phenotype of mice in which tanycytes of the arcuate nucleus and median eminence were conditionally ablated in adult mice. Although the cerebrospinal fluid-hypothalamic barrier was rendered more permeable following tanycyte ablation, neither the blood-hypothalamic barrier nor leptin-induced pSTAT3 activation in hypothalamic parenchyma were affected. We observed a significant increase in visceral fat distribution accompanying insulin insensitivity in male mice, without significant effect on either body weight or food intake. A high-fat diet tended to accelerate overall body weight gain in tanycyte-ablated mice, but the development of visceral adiposity and insulin insensitivity was comparable to wildtype. Thermoneutral housing exacerbated fat accumulation and produced a shift away from fat oxidation in tanycyte-ablated mice. These results clarify the extent to which tanycytes regulate energy balance, and demonstrate a role for tanycytes in regulating fat metabolism.
Asunto(s)
Tejido Adiposo/metabolismo , Núcleo Arqueado del Hipotálamo/metabolismo , Células Ependimogliales/metabolismo , Eliminación de Gen , Eminencia Media/metabolismo , Obesidad/metabolismo , Animales , Núcleo Arqueado del Hipotálamo/química , Metabolismo Energético/fisiología , Células Ependimogliales/química , Masculino , Eminencia Media/química , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/genéticaRESUMEN
The bidirectional water channel aquaporin 4 (AQP4) is abundantly expressed in the neural tissue. The advantages and disadvantages of AQP4 neural tissue deficiency under pathological conditions, such as inflammation, and relationship with neural diseases, such as Alzheimer's disease, have been previously reported. However, the physiological functions of AQP4 are not fully understood. Here, we evaluated the role of AQP4 in the mouse retina using Aqp4 knockout (KO) mice. Aqp4 was expressed in Müller glial cells surrounding the synaptic area between photoreceptors and bipolar cells. Both scotopic and photopic electroretinograms showed hyperactive visual responses in KO mice, gradually progressing with age. Moreover, the amplitude reduction after frequent stimuli and synaptic fatigue was more severe in KO mice. Glutamine synthetase, glutamate aspartate transporter, synaptophysin, and the inward potassium channel Kir2.1, but not Kir4.1, were downregulated in KO retinas. KIR2.1 colocalized with AQP4 in Müller glial cells at the synaptic area, and its expression was affected by Aqp4 levels in primary Müller glial cell cultures. Intraocular injection of potassium in wild-type mice led to visual function hyperactivity, as observed in Aqp4 KO mice. Mitochondria molecules, such as Pgc1α and CoxIV, were downregulated, while apoptotic markers were upregulated in KO retinas. AQP4 may fine-tune synaptic activity, most likely by regulating potassium metabolism, at least in part, via collaborating with KIR2.1, and possibly indirectly regulating glutamate kinetics, to inhibit neural hyperactivity and synaptic fatigue which finally affect mitochondria and cause neurodegeneration.
Asunto(s)
Acuaporina 4/metabolismo , Retina/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Visión Ocular/fisiología , Animales , Acuaporina 4/análisis , Células Cultivadas , Células Ependimogliales/química , Células Ependimogliales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canales de Potasio de Rectificación Interna/análisis , Canales de Potasio de Rectificación Interna/metabolismo , Retina/química , Sinapsis/químicaRESUMEN
Neuronal migration is essential for the orchestration of brain development and involves several contiguous steps: interkinetic nuclear movement (INM), multipolar-bipolar transition, locomotion, and translocation. Growing evidence suggests that Rho GTPases, including RhoA, Rac, Cdc42, and the atypical Rnd members, play critical roles in neuronal migration by regulating both actin and microtubule cytoskeletal components. This review focuses on the spatiotemporal-specific regulation of Rho GTPases as well as their regulators and effectors in distinct steps during the neuronal migration process. Their roles in bridging extracellular signals and cytoskeletal dynamics to provide optimal structural support to the migrating neurons will also be discussed.
Asunto(s)
Neuronas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Uniones Adherentes/metabolismo , Animales , Movimiento Celular , Células Ependimogliales/química , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Humanos , Neurogénesis , Neuronas/citología , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rho/antagonistas & inhibidoresRESUMEN
The retina is one of the most metabolically active tissues in the body and utilizes glucose to produce energy and intermediates required for daily renewal of photoreceptor cell outer segments. Glucose transporter 1 (GLUT1) facilitates glucose transport across outer blood retinal barrier (BRB) formed by the retinal pigment epithelium (RPE) and the inner BRB formed by the endothelium. We used conditional knockout mice to study the impact of reducing glucose transport across the RPE on photoreceptor and Müller glial cells. Transgenic mice expressing Cre recombinase under control of the Bestrophin1 ( Best1) promoter were bred with Glut1flox/flox mice to generate Tg-Best1-Cre:Glut1flox/flox mice ( RPEΔGlut1). The RPEΔGlut1 mice displayed a mosaic pattern of Cre expression within the RPE that allowed us to analyze mice with ~50% ( RPEΔGlut1m) recombination and mice with >70% ( RPEΔGlut1h) recombination separately. Deletion of GLUT1 from the RPE did not affect its carrier or barrier functions, indicating that the RPE utilizes other substrates to support its metabolic needs thereby sparing glucose for the outer retina. RPEΔGlut1m mice had normal retinal morphology, function, and no cell death; however, where GLUT1 was absent from a span of RPE greater than 100 µm, there was shortening of the photoreceptor cell outer segments. RPEΔGlut1h mice showed outer segment shortening, cell death of photoreceptors, and activation of Müller glial cells. The severe phenotype seen in RPEΔGlut1h mice indicates that glucose transport via the GLUT1 transporter in the RPE is required to meet the anabolic and catabolic requirements of photoreceptors and maintain Müller glial cells in a quiescent state.
Asunto(s)
Células Ependimogliales/metabolismo , Transportador de Glucosa de Tipo 1/biosíntesis , Glucosa/metabolismo , Células Fotorreceptoras/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Animales , Células Ependimogliales/química , Expresión Génica , Transportador de Glucosa de Tipo 1/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Células Fotorreceptoras/química , Epitelio Pigmentado de la Retina/químicaRESUMEN
The transient receptor potential vanilloid isoform 4 (TRPV4) functions as polymodal transducer of swelling, heat, stretch, and lipid metabolites, is widely expressed across sensory tissues, and has been implicated in pressure sensing in vertebrate retinas. Although TRPV4 knockout mice exhibit a variety of mechanosensory, nociceptive, and thermo- and osmoregulatory phenotypes, it is not known whether the transmission of light-induced signals in the eye is affected by the loss of TRPV4. We utilized field potentials, a measure of rod and cone signaling, to determine whether TRPV4 impacts on the generation and/or transmission of the photoreceptor light response and neurotransmission. Luminance intensity-response relationships were acquired in anesthetized wild-type and TRPV4-/- mice and evaluated for peak amplitude and implicit time under scotopic and photopic conditions. We found that the morphology of the outer retina is unaffected by the ablation of the Trpv4 gene. Calcium imaging of dissociated Müller glia showed that selective TRPV4 stimulation induces oscillatory calcium signals in adjacent rods. However, no differences in scotopic or photopic light-evoked signaling in the distal retina were observed in TRPV4-/- eyes, suggesting that TRPV4 signaling in healthy Müller cells does not modulate the transmission of light-evoked signals at rod and cone synapses.
Asunto(s)
Fototransducción/fisiología , Células Fotorreceptoras de Vertebrados/efectos de la radiación , Canales Catiónicos TRPV/fisiología , Animales , Señalización del Calcio/fisiología , Electrorretinografía , Células Ependimogliales/química , Células Ependimogliales/fisiología , Leucina/análogos & derivados , Leucina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Visión Nocturna/fisiología , Células Fotorreceptoras de Vertebrados/metabolismo , Células Ganglionares de la Retina/química , Células Ganglionares de la Retina/fisiología , Sulfonamidas/farmacología , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/deficiencia , Canales Catiónicos TRPV/genéticaRESUMEN
BACKGROUND: Imaging and histology of clear-cell ependymoma and cerebellum-based hemangioblastoma are similar; distinguishing between them is a diagnostic challenge. CASE PRESENTATION: A 62-year-old Chinese woman presented with an intermittent headache of 8 years' duration. Computed tomography and magnetic resonance imaging revealed a mass in the cerebellum. Neurological imaging suggested hemangioblastoma (HB). Histologically, the tumor included cellular and paucicellular areas, in which cells were arranged in nests or diffusely distributed; and a highly vascular area, in which tumor cells were arranged in clusters and separated by capillaries. At low magnification, the tumor mimicked cellular HB, but at high magnification, tumor cells showed clear cytoplasm instead of the vacuolated cytoplasm typically observed in HB. Moreover, spindly, bipolar elements resembling tanycytes were observed within the nest structures. Although these features indicated the possibility of ependymoma, neither true ependymal rosettes nor an ependymal-lined profile was observed. The tumor was characterized by prominent vascularity, but glomeruloid formation was absent. We saw pleomorphism in foci of some tumor giant cells, but pathologic mitosis and palisaded necrosis were absent. Most tumor cells were positive for glial fibrillary acidic protein and S100. Epithelial membrane antigen was expressed with a paranuclear dot-like or a ring-like pattern. The Ki-67 index was approximately 2%. Considering the patient's symptom, neurological imaging, and pathological findings, she was diagnosed as cerebellar ependymoma (WHO grade II). CONCLUSIONS: Here, we report a case of ependymoma with overlapping clear-cell and tanycytic features, and review the literature to evaluate its real incidence. Pathologists should consider this rare diagnosis when confronted with a similar presentation.
Asunto(s)
Neoplasias Cerebelosas/patología , Células Ependimogliales/patología , Ependimoma/patología , Hemangioblastoma/patología , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Biopsia , Neoplasias Cerebelosas/química , Neoplasias Cerebelosas/genética , Angiografía Cerebral/métodos , Deleción Cromosómica , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 19 , Angiografía por Tomografía Computarizada , Diagnóstico Diferencial , Células Ependimogliales/química , Ependimoma/química , Ependimoma/genética , Femenino , Hemangioblastoma/química , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Persona de Mediana Edad , Clasificación del Tumor , Valor Predictivo de las PruebasRESUMEN
PURPOSE: To investigate associations between diabetic macular edema (DME) and levels of the Wnt modulator Dickkopf 3 (DKK-3) in the aqueous humor (AH) of patients with DME and to analyze the clinical implications of this association. METHODS: Forty-four eyes of 39 patients with DME and 27 eyes of 27 controls were studied. Aqueous humor DKK-3 levels were measured by ELISA before the first intravitreal injection of bevacizumab (IVB). Visual acuity assessments and spectral-domain optical coherence tomography (SD-OCT) were performed before and 3 months after the first IVB. DKK-3 expression in high glucose-treated human Müller cells was examined by Western blot and immunofluorescence. Concentration of secreted DKK-3 in conditioned medium from human Müller cell cultures were analyzed by ELISA. RESULTS: ELISA showed increased DKK-3 levels in the eyes of DME patients compared with control subjects (median 207.86 ng/mL, range, 66.75-499.64 vs. 94.94 ng/mL, 33.34-164.45 ng/mL; P < 0.001). Based on multivariate analyses, elevated DKK-3 levels were associated with increased inner nuclear layer (INL) volume on SD-OCT before IVB. Western blot and immunofluorescence analyses showed higher DKK-3 expression in high glucose-treated Müller cells than in control cells, with DKK-3 secretion also being increased. CONCLUSIONS: DKK-3 expression was elevated in the AH of DME patients and in high glucose-treated human Müller cells. The observation of increased DKK-3 expression levels in the AH of DME patients with prominent edema in the INL suggests that the area of INL thickening might correlate with the area of reactive responses by Müller cells in these patients.
Asunto(s)
Humor Acuoso/química , Complicaciones de la Diabetes/metabolismo , Péptidos y Proteínas de Señalización Intercelular/análisis , Edema Macular/etiología , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Quimiocinas , Ensayo de Inmunoadsorción Enzimática , Células Ependimogliales/química , Células Ependimogliales/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Edema Macular/metabolismo , Masculino , Persona de Mediana Edad , Tomografía de Coherencia Óptica , Agudeza VisualRESUMEN
Specialized intermediate filaments (IFs) have critical importance for the clearness and uncommon transparency of vertebrate lens fiber cells, although the physical mechanisms involved are poorly understood. Recently, an unusual low-scattering light transport was also described in retinal Müller cells. Exploring the function of IFs in Müller cells, we have studied the morphology and distribution pattern of IFs and other cytoskeletal filaments inside the Müller cell main processes in the foveolar part of the avian (pied flycatcher) retina. We found that some IFs surrounded by globular nanoparticles (that we suggest are crystallines) are present in almost every part of the Müller cells that span the retina, including the microvilli. Unlike IFs implicated in the mechanical architecture of the cell, these IFs are not connected to any specific cellular membranes. Instead, they are organized into bundles, passing inside the cell from the endfeet to the photoreceptor, following the geometry of the processes, and repeatedly circumventing numerous obstacles. We believe that the presently reported data effectively confirm that the model of nanooptical channels built of the IFs may provide a viable explanation of Müller cell transparency.
Asunto(s)
Células Ependimogliales/ultraestructura , Filamentos Intermedios/ultraestructura , Retina/citología , Pájaros Cantores/anatomía & histología , Animales , Fenómenos Biofísicos , Células Ependimogliales/química , Células Ependimogliales/fisiología , Retina/fisiología , Pájaros Cantores/fisiologíaRESUMEN
Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms. There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.
Asunto(s)
Barrera Hematorretinal/fisiopatología , Dependovirus/fisiología , Enfermedades de la Retina/fisiopatología , Animales , Barrera Hematorretinal/patología , Barrera Hematorretinal/virología , Permeabilidad Capilar , Dependovirus/genética , Células Ependimogliales/química , Células Ependimogliales/patología , Células Ependimogliales/virología , Técnicas de Transferencia de Gen , Genes Reporteros , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Pericitos/química , Pericitos/patología , Pericitos/virología , Enfermedades de la Retina/patología , Enfermedades de la Retina/virología , TransfecciónRESUMEN
OBJECTIVE: Neovascularization and vaso-obliteration are vision-threatening events that develop by interactions between retinal vascular and glial cells. A high-salt diet is causal in cardiovascular and renal disease, which is linked to modulation of the renin-angiotensin-aldosterone system. However, it is not known whether dietary salt influences retinal vasculopathy and if the renin-angiotensin-aldosterone system is involved. We examined whether a low-salt (LS) diet influenced vascular and glial cell injury and the renin-angiotensin-aldosterone system in ischemic retinopathy. APPROACH AND RESULTS: Pregnant Sprague Dawley rats were fed LS (0.03% NaCl) or normal salt (0.3% NaCl) diets, and ischemic retinopathy was induced in the offspring. An LS diet reduced retinal neovascularization and vaso-obliteration, the mRNA and protein levels of the angiogenic factors, vascular endothelial growth factor, and erythropoietin. Microglia, which influence vascular remodeling in ischemic retinopathy, were reduced by LS as was tumor necrosis factor-α. Macroglial Müller cells maintain the integrity of the blood-retinal barrier, and in ischemic retinopathy, LS reduced their gliosis and also vascular leakage. In retina, LS reduced mineralocorticoid receptor, angiotensin type 1 receptor, and renin mRNA levels, whereas, as expected, plasma levels of aldosterone and renin were increased. The aldosterone/mineralocorticoid receptor-sensitive epithelial sodium channel alpha (ENaCα), which is expressed in Müller cells, was increased in ischemic retinopathy and reduced by LS. In cultured Müller cells, high salt increased ENaCα, which was prevented by mineralocorticoid receptor and angiotensin type 1 receptor blockade. Conversely, LS reduced ENaCα, angiotensin type 1 receptor, and mineralocorticoid receptor expression. CONCLUSIONS: An LS diet reduced retinal vasculopathy, by modulating glial cell function and the retinal renin-angiotensin-aldosterone system.
Asunto(s)
Dieta Hiposódica , Canales Epiteliales de Sodio/fisiología , Microglía/fisiología , Sistema Renina-Angiotensina/fisiología , Neovascularización Retiniana/dietoterapia , Complejo 1 de Proteína Adaptadora/análisis , Aldosterona/sangre , Aldosterona/fisiología , Animales , Animales Recién Nacidos , Acuaporina 4/biosíntesis , Acuaporina 4/genética , Peso Corporal , Células Cultivadas , Modelos Animales de Enfermedad , Conducta de Ingestión de Líquido , Células Ependimogliales/química , Células Ependimogliales/patología , Eritropoyetina/análisis , Gliosis/etiología , Gliosis/fisiopatología , Hematócrito , Transporte Iónico , Isquemia/fisiopatología , Glomérulos Renales/patología , Sistema de Señalización de MAP Quinasas , Fosforilación , Canales de Potasio de Rectificación Interna/biosíntesis , Canales de Potasio de Rectificación Interna/genética , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Células Ganglionares de la Retina/metabolismo , Neovascularización Retiniana/fisiopatología , Neovascularización Retiniana/prevención & control , Retinopatía de la Prematuridad , Sodio/metabolismo , Cloruro de Sodio Dietético/efectos adversos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
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 injury-dependent 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.