RESUMEN
More than twelve morphologically and physiologically distinct subtypes of primary somatosensory neuron report salient features of our internal and external environments1-4. It is unclear how specialized gene expression programs emerge during development to endow these subtypes with their unique properties. To assess the developmental progression of transcriptional maturation of each subtype of principal somatosensory neuron, we generated a transcriptomic atlas of cells traversing the primary somatosensory neuron lineage in mice. Here we show that somatosensory neurogenesis gives rise to neurons in a transcriptionally unspecialized state, characterized by co-expression of transcription factors that become restricted to select subtypes as development proceeds. Single-cell transcriptomic analyses of sensory neurons from mutant mice lacking transcription factors suggest that these broad-to-restricted transcription factors coordinate subtype-specific gene expression programs in subtypes in which their expression is maintained. We also show that neuronal targets are involved in this process; disruption of the prototypic target-derived neurotrophic factor NGF leads to aberrant subtype-restricted patterns of transcription factor expression. Our findings support a model in which cues that emanate from intermediate and final target fields promote neuronal diversification in part by transitioning cells from a transcriptionally unspecialized state to transcriptionally distinct subtypes by modulating the selection of subtype-restricted transcription factors.
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Neurogénesis , Neuronas/fisiología , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Factor de Crecimiento Nervioso/metabolismo , Neuronas/citología , ARN/análisis , ARN/genética , Análisis de la Célula Individual , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/metabolismo , Factor de Transcripción Brn-3C/genética , Factor de Transcripción Brn-3C/metabolismoRESUMEN
Retinal ganglion cell (RGC) degeneration is a hallmark of glaucoma, the most prevalent cause of irreversible blindness. Thus, therapeutic strategies are needed to protect and replace these projection neurons. One innovative approach is to promote de novo genesis of RGCs via manipulation of endogenous cell sources. Here, we demonstrate that the pluripotency regulator gene Krüppel-like factor 4 (Klf4) is sufficient to change the potency of lineage-restricted retinal progenitor cells to generate RGCs in vivo Transcriptome analysis disclosed that the overexpression of Klf4 induces crucial regulators of RGC competence and specification, including Atoh7 and Eya2 In contrast, loss-of-function studies in mice and zebrafish demonstrated that Klf4 is not essential for generation or differentiation of RGCs during retinogenesis. Nevertheless, induced RGCs (iRGCs) generated upon Klf4 overexpression migrate to the proper layer and project axons aligned with endogenous fascicles that reach the optic nerve head. Notably, iRGCs survive for up to 30â days after in vivo generation. We identified Klf4 as a promising candidate for reprogramming retinal cells and regenerating RGCs in the retina.This article has an associated 'The people behind the papers' interview.
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Factores de Transcripción de Tipo Kruppel/fisiología , Neurogénesis , Células Ganglionares de la Retina/fisiología , Animales , Ciclo Celular , Femenino , Proteínas de Homeodominio/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regeneración Nerviosa , Células-Madre Neurales/fisiología , Ratas , Factor de Transcripción Brn-3A/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Pez Cebra , Proteínas de Pez Cebra/fisiologíaRESUMEN
BACKGROUND: Bladder cancer is one of the most common malignancies but the corresponding diagnostic methods are either invasive or limited in specificity and/or sensitivity. This study aimed to develop a urine-based methylation panel for bladder cancer detection by improving published panels and validate performance of the new panel with clinical samples. METHODS: Related researches were reviewed and 19 potential panels were selected. RRBS was performed on a cohort with 45 samples to reassess these panels and a new panel inherited best markers was developed. The new panel was applied with qMSP platform to 33 samples from the RRBS cohort and the results were compared to those of RRBS. Lastly, another larger cohort with 207 samples was used to validate new panel performance with qMSP. RESULTS: Three biomarkers (PCDH17, POU4F2 and PENK) were selected to construct a new panel P3. P3 panel achieved 100% specificity and 71% sensitivity with RRBS in corresponding cohort and then showed a better performance of 100% specificity and 84% sensitivity with qMSP platforms in a balanced cohort. When validated with 207-sample cohort, P3 with qMSP showed a performance of 97% specificity and 87% sensitivity which was modestly improved compared to the panels it derided from. CONCLUSIONS: Overall, the P3 panel achieved relatively high sensitivity and accuracy in bladder cancer detection.
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Metilación de ADN , Detección Precoz del Cáncer/métodos , Urinálisis/métodos , Neoplasias de la Vejiga Urinaria/diagnóstico , Orina/química , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/orina , Cadherinas/orina , Encefalinas/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Precursores de Proteínas/orina , Sensibilidad y Especificidad , Factor de Transcripción Brn-3B/orinaRESUMEN
The melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) are a relatively recently discovered class of atypical ganglion cell photoreceptor. These ipRGCs are a morphologically and physiologically heterogeneous population that project widely throughout the brain and mediate a wide array of visual functions ranging from photoentrainment of our circadian rhythms, to driving the pupillary light reflex to improve visual function, to modulating our mood, alertness, learning, sleep/wakefulness, regulation of body temperature, and even our visual perception. The presence of melanopsin as a unique molecular signature of ipRGCs has allowed for the development of a vast array of molecular and genetic tools to study ipRGC circuits. Given the emerging complexity of this system, this review will provide an overview of the genetic tools and methods used to study ipRGCs, how these tools have been used to dissect their role in a variety of visual circuits and behaviors in mice, and identify important directions for future study.
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Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Opsinas de Bastones/metabolismo , Animales , Animales Modificados Genéticamente/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/ultraestructura , Opsinas de Bastones/genética , Canales Catiónicos TRPC/metabolismo , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/metabolismo , Fosfolipasas de Tipo C/metabolismo , Vías Visuales/fisiologíaRESUMEN
As a POU homeodomain transcription factor, POU4F2 has been implicated in regulating tumorigenic processes in various cancers. However, the role of POU4F2 in colorectal cancer (CRC) remains unclear. Here, we revealed that POU4F2 functions as a tumor promotor in CRC. Bioinformatics analysis in specimens from CRC patients and expression analysis in CRC cell lines showed that POU4F2 was upregulated at the mRNA and protein levels in CRC. Depletion of POU4F2 suppressed the metastatic phenotypes of CRC cells, including cell migration, invasion, and the expression of epithelial-mesenchymal transition (EMT) markers. Moreover, depletion of POU4F2 decreased the number of lung metastatic nodes in nude mice. Mechanistically, POU4F2 positively regulated the Hedgehog signaling pathway, as inferred from the downregulation of the expression of sonic Hedgehog homolog, patched 1, Smoothened, and GLI family zinc finger 1 in vitro and vivo following silencing of POU4F2. Furthermore, the SMO agonist SAG reversed the effects of POU4F2 knockdown in CRC. Functionally, POU4F2 contributed to the Hedgehog signaling-regulated activation of the EMT process and promotion of CRC cell migration and invasion. Collectively, these findings elucidated the role of POU4F2 as a tumor promotor in CRC through the regulation of Hedgehog signaling-mediated EMT and suggested that POU4F2 suppression might be a promising therapeutic target in inhibiting CRC metastasis.
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Movimiento Celular , Neoplasias Colorrectales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Proteínas Hedgehog/metabolismo , Invasividad Neoplásica , Factor de Transcripción Brn-3B/fisiología , Animales , Línea Celular Tumoral , Colon/metabolismo , Colon/patología , Neoplasias Colorrectales/patología , Ciclohexilaminas/farmacología , Regulación hacia Abajo , Silenciador del Gen , Humanos , Neoplasias Pulmonares/secundario , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Receptor Patched-1/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Receptor Smoothened/agonistas , Receptor Smoothened/metabolismo , Tiofenos/farmacología , Factor de Transcripción Brn-3B/antagonistas & inhibidores , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/metabolismo , Regulación hacia Arriba , Dedos de ZincRESUMEN
Glaucoma is the second leading cause of blindness worldwide. This multifactorial, neurodegenerative group of diseases is characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, leading to irreversible visual impairment and blindness. There is a huge unmet and urging need for the development of new and translatable strategies and treatment options to prevent this progressive loss of RGC. Accumulating evidence points towards a critical role of neuroinflammation, in particular microglial cells, in the pathogenesis of glaucoma. Leukotrienes are mediators of neuroinflammation and are involved in many neurodegenerative diseases. Therefore, we tested the leukotriene receptors CysLT1R/GPR17-selective antagonist Montelukast (MTK) for its efficacy to modulate the reactive state of microglia in order to ameliorate RGCs loss in experimental glaucoma. Ocular hypertension (OHT) was induced unilaterally by injection of 8 µm magnetic microbead (MB) into the anterior chamber of female Brown Norway rats. The contralateral, untreated eye served as control. Successful induction of OHT was verified by daily IOP measurement using a TonoLab rebound tonometer. Simultaneously to OHT induction, one group received daily MTK treatment and the control group vehicle solution by oral gavage. Animals were sacrificed 13-15 days after MB injection. Retina and optic nerves (ON) of OHT and contralateral eyes were analyzed by immunofluorescence with specific markers for RGCs (Brn3a), microglial cells/macrophages (Iba1 and CD68), and cysteinyl leukotriene pathway receptors (CysLT1R and GPR17). Protein labeling was documented by confocal microscopy and analyzed with ImageJ plugins. Further, mRNA expression of genes of the inflammatory and leukotriene pathway was analyzed in retinal tissue. MTK treatment resulted in a short-term IOP reduction at day 2, which dissipated by day 5 of OHT induction in MTK treated animals. Furthermore, MTK treatment resulted in a decreased activation of Iba1+ microglial cells in the retina and ON, and in a significantly increased RGC survival in OHT eyes. Within the retina, GPR17 and CysLT1R expression was demonstrated in single RCGs and in microglial cells respectively. Further, increased mRNA expression of pro-inflammatory genes was detected in OHT induced retinas. In the ON, OHT induction increased the number of GPR17+ cells, showing a trend of reduction following MTK treatment. This study shows for the first time a significantly increased RGC survival in an acute OHT model following treatment with the leukotriene receptor antagonist MTK. These results strongly suggest a neuroprotective effect of MTK and a potential new therapeutic strategy for glaucoma treatment.
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Antagonistas de Leucotrieno/uso terapéutico , Microglía/metabolismo , Hipertensión Ocular/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores de Leucotrienos/metabolismo , Células Ganglionares de la Retina/fisiología , Acetatos/uso terapéutico , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Biomarcadores/metabolismo , Proteínas de Unión al Calcio/metabolismo , Supervivencia Celular/fisiología , Ciclopropanos/uso terapéutico , Modelos Animales de Enfermedad , Electrorretinografía , Femenino , Regulación de la Expresión Génica/fisiología , Presión Intraocular/fisiología , Proteínas de Microfilamentos/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Hipertensión Ocular/fisiopatología , Quinolinas/uso terapéutico , ARN Mensajero/genética , Ratas , Ratas Endogámicas BN , Reacción en Cadena en Tiempo Real de la Polimerasa , Retina/metabolismo , Retina/fisiopatología , Sulfuros/uso terapéutico , Tonometría Ocular , Factor de Transcripción Brn-3B/metabolismoRESUMEN
BACKGROUND: During development, all retinal cell types arise from retinal progenitor cells (RPCs) in a step-wise fashion. Atoh7 and Pou4f2 mark, and function in, two phases of retinal ganglion cell (RGC) genesis; Atoh7 functions in a subpopulation of RPCs to render them competent for the RGC fate, whereas Pou4f2 participates in RGC fate specification and RGC differentiation. Despite extensive research on their roles, the properties of the two phases represented by these two factors have not been well studied, likely due to the retinal cellular heterogeneity. RESULTS: In this report, we describe two novel knock-in mouse alleles, Atoh7zsGreenCreERT2 and Pou4f2FlagtdTomato , which labeled retinal cells in the two phases of RGC development by fluorescent proteins. Also, the Atoh7zsGreenCreERT2 allele allowed for indirect labeling of RGCs and other cell types upon tamoxifen induction in a dose-dependent manner. Further, these alleles could be used to purify retinal cells in the different phases by fluorescence assisted cell sorting (FACS). Single cell RNA-seq analysis of purified cells from Atoh7zsGreenCreERT2 retinas further validated that this allele labeled both transitional/competent RPCs and their progenies including RGCs. CONCLUSIONS: Thus, these two alleles are very useful tools for studying the molecular and genetic mechanisms underlying RGC formation.
Asunto(s)
Alelos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Retina/embriología , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/metabolismo , Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismoRESUMEN
A variety of visual cues can trigger defensive reactions in mice and other species. In mice, looming stimuli that mimic an approaching aerial predator elicit flight or freezing reactions, while sweeping stimuli that mimic an aerial predator flying parallel to the ground typically elicit freezing. The retinal ganglion cell (RGC) types involved in these circuits are largely unknown. We previously discovered that loss of RGC subpopulations in Brn3b knockout mice results in distinct visual response deficits. Here, we report that retinal or global loss of Brn3b selectively ablates the fleeing response to looming stimuli while leaving the freeze response intact. In contrast, freezing responses to sweeping stimuli are significantly affected. Genetic manipulations removing three RGC subpopulations (Brn3a+ betta RGCs, Opn4+Brn3b+, and Brn3c+Brn3b+ RGCs) result in milder phenocopies of Brn3b knockout response deficits. These findings show that flight and freezing responses to distinct visual cues are mediated by circuits that can already be separated at the level of the retina, potentially by enlisting dedicated RGC types.NEW & NOTEWORTHY Flight and freezing response choices evoked by visual stimuli are controlled by brain stem and thalamic circuits. Genetically modified mice with loss of specific retinal ganglion cell (RGC) subpopulations have altered flight versus freezing choices in response to some but not other visual stimuli. This finding suggests that "threatening" visual stimuli may be computed already at the level of the retina and communicated via dedicated pathways (RGCs) to the brain.
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Reacción de Prevención/fisiología , Células Ganglionares de la Retina/fisiología , Percepción Visual/fisiología , Animales , Conducta Animal , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción Brn-3B/genética , Factor de Transcripción Brn-3B/fisiologíaRESUMEN
Regulated retinal ganglion cell (RGC) differentiation and axonal guidance is required for a functional visual system. Homeodomain and basic helix-loop-helix transcription factors are required for retinogenesis, as well as patterning, differentiation and maintenance of specific retinal cell types. We hypothesized that Dlx1, Dlx2 and Brn3b homeobox genes function in parallel intrinsic pathways to determine RGC fate and therefore generated Dlx1/Dlx2/Brn3b triple-knockout mice. A more severe retinal phenotype was found in the Dlx1/Dlx2/Brn3b-null retinas than was predicted by combining features of the Brn3b single- and Dlx1/Dlx2 double-knockout retinas, including near total RGC loss with a marked increase in amacrine cells in the ganglion cell layer. Furthermore, we discovered that DLX1 and DLX2 function as direct transcriptional activators of Brn3b expression. Knockdown of Dlx2 expression in primary embryonic retinal cultures and Dlx2 gain of function in utero strongly support that DLX2 is both necessary and sufficient for Brn3b expression in vivo We suggest that ATOH7 specifies RGC-committed progenitors and that Dlx1 and Dlx2 function both downstream of ATOH7 and in parallel, but cooperative, pathways that involve regulation of Brn3b expression to determine RGC fate.
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Diferenciación Celular , Proteínas de Homeodominio/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Factores de Transcripción/metabolismo , Vertebrados/metabolismo , Células Amacrinas/citología , Células Amacrinas/metabolismo , Animales , Apoptosis/genética , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Recuento de Células , División Celular/genética , Linaje de la Célula/genética , Proliferación Celular , Células Cultivadas , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/metabolismo , Electroporación , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones Noqueados , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción Brn-3B/deficiencia , Factores de Transcripción/deficienciaRESUMEN
Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a Brn3bCre knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in Vegfa120/120 and Vegfa188/188 mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and Vegfa188/188 mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.
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Axones/metabolismo , Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Neuropilina-1/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Tipificación del Cuerpo , Diencéfalo/embriología , Diencéfalo/metabolismo , Células Endoteliales/metabolismo , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/metabolismo , Ratones Endogámicos C57BL , Mutación/genética , Neovascularización Fisiológica , Quiasma Óptico/embriología , Quiasma Óptico/metabolismo , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Vías Visuales/metabolismoRESUMEN
Purpose: The present study aimed to determine whether the administration of Acer palmatum thumb. leaf extract (KIOM-2015E) protects against the degeneration of rat retinal ganglion cells after ischemia/reperfusion (I/R) induced by midbrain cerebral artery occlusion (MCAO). Methods: Sprague-Dawley rats were subjected to 90 min of MCAO, which produces transient ischemia in both the retina and brain due to the use of an intraluminal filament that blocks the ophthalmic and middle cerebral arteries. This was followed by reperfusion under anesthesia with isoflurane. The day after surgery, the eyes were treated three times (eye drop) or one time (oral administration) daily with KIOM-2015E for five days. Retinal histology was assessed in flat mounts and vertical sections to determine the effect of KIOM-2015E on I/R injury. Results: A significant loss of brain-specific homeobox/POU domain protein 3A (Brn3a) and neuron-specific class III beta-tubulin (Tuj-1) fluorescence and a marked increase in glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS) expression were observed after five days in the PBS-treated MCAO group compared to the sham-operated control group. However, KIOM-2015E treatment reduced (1) MCAO-induced upregulation of GFAP and GS, (2) retinal ganglion cell loss, (3) nerve fiber degeneration, and (4) the number of TUNEL-positive cells. KIOM-2015E application also increased staining for parvalbumin (a marker of horizontal cell associated calcium-binding protein and amacrine cells) and recoverin (a marker of photoreceptor expression) in rats subjected to MCAO-induced retinal damage. Conclusions: Our findings indicated that KIOM-2015E treatment exerted protective effects against retinal damage following MCAO injury and that this extract may aid in the development of novel therapeutic strategies for retinal diseases, such as glaucoma and age-related macular disease.
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Acer/metabolismo , Apoptosis/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Daño por Reperfusión/metabolismo , Degeneración Retiniana/prevención & control , Células Ganglionares de la Retina/efectos de los fármacos , Acer/química , Animales , Cromatografía Líquida de Alta Presión , Regulación hacia Abajo , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Masculino , Fibras Nerviosas/patología , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/complicaciones , Daño por Reperfusión/mortalidad , Degeneración Retiniana/complicaciones , Degeneración Retiniana/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/patología , Factor de Transcripción Brn-3B/metabolismo , Tubulina (Proteína)/metabolismo , Regulación hacia ArribaRESUMEN
Brn3b (Pou4f2) is a class-4 POU domain transcription factor known to play central roles in the development of different neuronal populations of the Central Nervous System, including retinal ganglion cells (RGCs), the neurons that connect the retina with the visual centers of the brain. Here, we have used CRISPR-based genetic engineering to generate a Brn3b-mCherry reporter mouse without altering the endogenous expression of Brn3b. In our mouse line, mCherry faithfully recapitulates normal Brn3b expression in the retina, the optic tracts, the midbrain tectum, and the trigeminal ganglia. The high sensitivity of mCherry also revealed novel expression of Brn3b in the neuroectodermal cells of the optic stalk during early stages of eye development. Importantly, the fluorescent intensity of Brn3b-mCherry in our reporter mice allows for noninvasive live imaging of RGCs using Scanning Laser Ophthalmoscopy (SLO), providing a novel tool for longitudinal monitoring of RGCs.
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Proteínas de Homeodominio/genética , Proteínas Luminiscentes/metabolismo , Retina/metabolismo , Factor de Transcripción Brn-3B/genética , Animales , Sistemas CRISPR-Cas , Genes Reporteros , Proteínas de Homeodominio/metabolismo , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retina/diagnóstico por imagen , Factor de Transcripción Brn-3B/metabolismo , Vías Visuales/diagnóstico por imagen , Vías Visuales/metabolismo , Proteína Fluorescente RojaRESUMEN
As with other retinal cell types, retinal ganglion cells (RGCs) arise from multipotent retinal progenitor cells (RPCs), and their formation is regulated by a hierarchical gene-regulatory network (GRN). Within this GRN, three transcription factors--atonal homolog 7 (Atoh7), POU domain, class 4, transcription factor 2 (Pou4f2), and insulin gene enhancer protein 1 (Isl1)--occupy key node positions at two different stages of RGC development. Atoh7 is upstream and is required for RPCs to gain competence for an RGC fate, whereas Pou4f2 and Isl1 are downstream and regulate RGC differentiation. However, the genetic and molecular basis for the specification of the RGC fate, a key step in RGC development, remains unclear. Here we report that ectopic expression of Pou4f2 and Isl1 in the Atoh7-null retina using a binary knockin-transgenic system is sufficient for the specification of the RGC fate. The RGCs thus formed are largely normal in gene expression, survive to postnatal stages, and are physiologically functional. Our results indicate that Pou4f2 and Isl1 compose a minimally sufficient regulatory core for the RGC fate. We further conclude that during development a core group of limited transcription factors, including Pou4f2 and Isl1, function downstream of Atoh7 to determine the RGC fate and initiate RGC differentiation.
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Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Factores de Transcripción/metabolismo , Alelos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ciclo Celular , Diferenciación Celular , Sistema Nervioso Central/metabolismo , Electrofisiología , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Retina/embriología , Retina/metabolismo , Células Madre/citologíaRESUMEN
Retinal ganglion cells (RGCs) are projection neurons in the neural retina that relay visual information from the environment to the central nervous system. The early expression of MATH5 endows the post-mitotic precursors with RGC competence and leads to the activation ofBrn3bthat marks committed RGCs. Nevertheless, this fate commitment process and, specifically, regulation ofBrn3bremain elusive. To explore the molecular mechanisms underlying RGC generation in the mouse retina, we analyzed the expression and function of Fez family zinc finger 2 (FEZF2), a transcription factor critical for the development of projection neurons in the cerebral cortex.Fezf2mRNA and protein were transiently expressed at embryonic day 16.5 in the inner neuroblast layer and the prospective ganglion cell layer of the retina, respectively. Knockout ofFezf2in the developing retina reduced BRN3B+ cells and increased apoptotic cell markers.Fezf2knockdown by retinalin uteroelectroporation diminished BRN3B but not the coexpressed ISLET1 and BRN3A, indicating that the BRN3B decrease was the cause, not the result, of the overall reduction of BRN3B+ RGCs in theFezf2knockout retina. Moreover, the mRNA and promoter activity ofBrn3bwere increasedin vitroby FEZF2, which bound to a 5' regulatory fragment in theBrn3bgenomic locus. These results indicate that transient expression ofFezf2in the retina modulates the transcription ofBrn3band the survival of RGCs. This study improves our understanding of the transcriptional cascade required for the specification of RGCs and provides novel insights into the molecular basis of retinal development.
Asunto(s)
Apoptosis/fisiología , Proteínas de Unión al ADN/biosíntesis , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/biosíntesis , Transcripción Genética/fisiología , Animales , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Proteínas de Homeodominio/genética , Ratones , Ratones Mutantes , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Células Ganglionares de la Retina/citología , Factor de Transcripción Brn-3B/genéticaRESUMEN
Retinal ganglion cell layer (RGCs) is one of the important layers of retina, depleted in Glaucoma. Loss of RGC neurons is a major cellular mechanism involved in its pathogenesis resulting in severe vision loss. Stem cell therapy has emerged as a potential strategy to arrest the apoptotic loss of RGCs and also replace the degenerative cells in damaged retina. Here, we have investigated the incorporation and survival of mouse bone marrow derived Lin-ve stem cells in N-methyl-d-aspartate (NMDA)-induced mouse model of retinal degeneration. Two days after intravitreal injection of NMDA (100 mM) showed significant decrease in ganglion cell number and increase in TUNEL positive apoptotic cells in retinal layers. The injury was further characterized by immunohistochemical expression of Brn3b, GFAP, Bcl2, pCREB, CNTF, GDNF, and BDNF in retinal layers. Lin-ve cells (100,000 dose) were intravitreally transplanted after 2 days of injury and evaluated after 7, 14, and 21 days of transplantation. Transplanted cells were found to have migrated from intravitreal space and incorporated into injured retina at 7, 14, and 21 days post-transplantation. At 21 days Brn3b, CNTF, and BDNF expression was found to be upregulated whereas GDNF was downregulated when compared to respective injury time points. Molecular data showed decrease in the expression of Brn3b, BDNF, CNTF, and GDNF post transplantation when compared with injury groups. This study reveals that Lin-ve stem cells may exert neuroprotective effect in damaged retina mediated by participation of neurotrophic factors induced by stem cell transplantation at the site of injury. J. Cell. Biochem. 118: 1699-1711, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Células de la Médula Ósea/citología , N-Metilaspartato/toxicidad , Factores de Crecimiento Nervioso/metabolismo , Degeneración Retiniana/inducido químicamente , Degeneración Retiniana/metabolismo , Células Madre/citología , Animales , Células de la Médula Ósea/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Ciliar/metabolismo , Modelos Animales de Enfermedad , Femenino , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Retina/efectos de los fármacos , Retina/patología , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Trasplante de Células Madre , Células Madre/fisiología , Factor de Transcripción Brn-3B/metabolismoRESUMEN
Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and regulate a wide array of light-dependent physiological processes. Genetic ablation of ipRGCs eliminates circadian photoentrainment and severely disrupts the pupillary light reflex (PLR). Here we show that ipRGCs consist of distinct subpopulations that differentially express the Brn3b transcription factor, and can be functionally distinguished. Brn3b-negative M1 ipRGCs innervate the suprachiasmatic nucleus (SCN) of the hypothalamus, whereas Brn3b-positive ipRGCs innervate all other known brain targets, including the olivary pretectal nucleus. Consistent with these innervation patterns, selective ablation of Brn3b-positive ipRGCs severely disrupts the PLR, but does not impair circadian photoentrainment. Thus, we find that molecularly distinct subpopulations of M1 ipRGCs, which are morphologically and electrophysiologically similar, innervate different brain regions to execute specific light-induced functions.
Asunto(s)
Ritmo Circadiano/fisiología , Ritmo Circadiano/efectos de la radiación , Reflejo Pupilar/fisiología , Reflejo Pupilar/efectos de la radiación , Células Ganglionares de la Retina/fisiología , Células Ganglionares de la Retina/efectos de la radiación , Animales , Ritmo Circadiano/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Modelos Neurológicos , Núcleo Olivar/metabolismo , Reflejo Pupilar/genética , Células Ganglionares de la Retina/citología , Opsinas de Bastones/genética , Opsinas de Bastones/metabolismo , Núcleo Supraquiasmático/metabolismo , Factor de Transcripción Brn-3B/deficiencia , Factor de Transcripción Brn-3B/metabolismoRESUMEN
Pou4f2 acts as a key node in the comprehensive and step-wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2-green fluorescent protein (GFP) fusion protein expressed in RGCs. Co-localization of POU4F2 and GFP in the retina and brain of Pou4f2-GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild-type mice, the expression patterns of POU4F2 and POU4F1 and the co-expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2-GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2-GFP/GFP homozygote and wild-type mice. These results demonstrated that the development of RGCs in Pou4f2-GFP/GFP homozygote mice was the same as in wild-type mice. Thus, the present Pou4f2-GFP knock-in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.
Asunto(s)
Redes Reguladoras de Genes/genética , Proteínas Fluorescentes Verdes/genética , Proteínas de Homeodominio/genética , Células Ganglionares de la Retina/metabolismo , Factor de Transcripción Brn-3B/genética , Animales , Axones/metabolismo , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Proteínas de Homeodominio/biosíntesis , Ratones , Retina/crecimiento & desarrollo , Retina/metabolismo , Factor de Transcripción Brn-3B/biosíntesisRESUMEN
The vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is the high affinity ligand of the transcription factor vitamin D receptor (VDR) and therefore a direct regulator of transcription. Transcriptome-wide analysis of THP-1 human monocytes had indicated more than 600 genes to be significantly (p<0.05) stimulated after 4h incubation with 1,25(OH)2D3, but only 67 of them where more than 1.5-fold up-regulated. These include the genes encoding for the transcription factors BCL6, NFE2, POU4F2 and ELF4, which are controlled by one or two VDR binding sites within their chromosomal domains. The latter are defined via DNA loop formation mediated by the transcription factor CTCF that is highly conserved in its genome-wide loci. We found BCL6 being most responsive to 1,25(OH)2D3 and selected it for further analysis. An incubation of THP-1 cells with 1,25(OH)2D3 for 24 h resulted in a significant (p<0.001) change in the mRNA expression of more than 1600 genes, of which 132 were at least 2-fold up-regulated. About half of the latter genes are secondary 1,25(OH)2D3 targets, since they do not carry any VDR binding site within their chromosomal domain. Chromatin immunoprecipitation sequencing datasets indicated that the majority of these domains contain a BCL6 binding site. We followed the secondary transcriptional response to 1,25(OH)2D3 for eight representative gene examples and confirmed the binding of CTCF and BCL6 to their respective chromosomal domains. In conclusion, our study indicated that in monocytes most of the physiological responses to 1,25(OH)2D3 involve the action of the transcription factor BCL6.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Subunidad p45 del Factor de Transcripción NF-E2/biosíntesis , Factor de Transcripción Brn-3B/biosíntesis , Factores de Transcripción/biosíntesis , Vitamina D/administración & dosificación , Sitios de Unión , Factor de Unión a CCCTC , Línea Celular , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Subunidad p45 del Factor de Transcripción NF-E2/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-6 , ARN Mensajero/biosíntesis , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor de Transcripción Brn-3B/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos , Vitamina D/análogos & derivadosRESUMEN
The POU4F2/Brn-3b transcription factor has been identified as a potentially novel regulator of key metabolic processes. Loss of this protein in Brn-3b knockout (KO) mice causes profound hyperglycemia and insulin resistance (IR), normally associated with type 2 diabetes (T2D), whereas Brn-3b is reduced in tissues taken from obese mice fed on high-fat diets (HFD), which also develop hyperglycemia and IR. Furthermore, studies in C2C12 myocytes show that Brn-3b mRNA and proteins are induced by glucose but inhibited by insulin, suggesting that this protein is itself highly regulated in responsive cells. Analysis of differential gene expression in skeletal muscle from Brn-3b KO mice showed changes in genes that are implicated in T2D such as increased glycogen synthase kinase-3ß and reduced GLUT4 glucose transporter. The GLUT4 gene promoter contains multiple Brn-3b binding sites and is directly transactivated by this transcription factor in cotransfection assays, whereas chromatin immunoprecipitation assays confirm that Brn-3b binds to this promoter in vivo. In addition, correlation between GLUT4 and Brn-3b in KO tissues or in C2C12 cells strongly supports a close association between Brn-3b levels and GLUT4 expression. Since Brn-3b is regulated by metabolites and insulin, this may provide a mechanism for controlling key genes that are required for normal metabolic processes in insulin-responsive tissues and its loss may contribute to abnormal glucose uptake.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodominio/genética , Hiperglucemia/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , ARN Mensajero/metabolismo , Factor de Transcripción Brn-3B/genética , Animales , Peso Corporal/genética , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ácido Glucárico/farmacología , Intolerancia a la Glucosa/genética , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Proteínas de Homeodominio/efectos de los fármacos , Proteínas de Homeodominio/metabolismo , Immunoblotting , Insulina/farmacología , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/efectos de los fármacos , Mutación , ARN Mensajero/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción Brn-3B/efectos de los fármacos , Factor de Transcripción Brn-3B/metabolismoRESUMEN
Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures.