RESUMEN
Neural stem cells (NSCs) differentiate into neuron-fated intermediate progenitor cells (IPCs) via cell division. Although differentiation from NSCs to IPCs is a discrete process, recent transcriptome analyses identified a continuous transcriptional trajectory during this process, raising the question of how to reconcile these contradictory observations. In mouse NSCs, Hes1 expression oscillates, regulating the oscillatory expression of the proneural gene Neurog2, while Hes1 expression disappears in IPCs. Thus, the transition from Hes1 oscillation to suppression is involved in the differentiation of NSCs to IPCs. Here, we found that Neurog2 oscillations induce the accumulation of Tbr2, which suppresses Hes1 expression, generating an IPC-like gene expression state in NSCs. In the absence of Tbr2, Hes1 expression is up-regulated, decreasing the formation of IPCs. These results indicate that the Neurog2-Tbr2 axis forms a continuous transcriptional trajectory to an IPC-like neurogenic state in NSCs, which then differentiate into IPCs via cell division.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Proteínas del Tejido Nervioso , Células-Madre Neurales , Neurogénesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Animales , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Ratones , Neurogénesis/genética , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción HES-1/metabolismo , Factor de Transcripción HES-1/genética , Neuronas/metabolismo , Neuronas/citología , Proteínas de Dominio T BoxRESUMEN
Human induced pluripotent stem cells (hiPSCs) can differentiate into neurons and glia via neural progenitor cells and are widely used for neurogenic studies. However, directly visualizing the transition status during the neural differentiation of live cells is difficult. Here, targeting NEUROG2 (NGN2) and TUBB3 as markers of neurogenic cells and neurons, respectively, we established TUBB3EGFP/NGN2TagRFP dual-reporter hiPSCs using CRISPR-Cas9 technology. We induced the differentiation of cortical neurons from dual-reporter hiPSCs, successfully visualizing cell-fate conversion in two-dimensional (2D) culture and 3D organoids. The reporter cells were used to monitor drug effects to enhance neural induction, responses to gene knockdown, transplantation to the embryonic mouse brain, and live imaging at single-cell resolution. Notably, the earliest REELIN-positive neurons showed a distinctive migration pattern, and their production was accelerated by HES1-function loss. Together, these results demonstrate the potential for dual-reporter hiPSCs in therapeutic neural regeneration strategies and studies on human cortical development.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células-Madre Neurales , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular/genética , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Neuronas , OrganoidesRESUMEN
Direct neuronal reprogramming is a promising strategy to generate various types of neurons that are, otherwise, inaccessible for researchers. However, the efficiency of neuronal conversion is highly dependent on the transcription factor used, the identity of the initial cells to convert, their species' background, and the neuronal subtype to which cells will convert. Regardless of these conditioning factors, the apoptotic regulator Bcl-2 acts as a pan-neuronal reprogramming enhancer. Bcl-2 mediates its effect in reprogramming by preventing an overshot of oxidative stress during the acquisition of a neuronal oxidative metabolism, thus reducing cell death by ferroptosis and facilitating the phenotypic conversion. In this chapter, we outline two methods to obtain either mouse or human neurons derived from postnatal astrocytes and skin fibroblasts, respectively. The overall reprogramming strategy is based on the co-expression of Bcl-2 and the transcription factor Neurog2 that produces mostly excitatory neurons. However, the method can be easily adapted to achieve alternative neuronal subtypes by using additional transcription factors, such as Isl1 for motor neurons. Therefore, our approaches provide solid but flexible platforms to obtain human and mouse induced neurons in vitro that can be applied to basic or translational research.
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Astrocitos/citología , Astrocitos/metabolismo , Técnicas de Reprogramación Celular , Reprogramación Celular/genética , Fibroblastos/citología , Neuronas/citología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Astrocitos/efectos de los fármacos , Técnicas de Cultivo de Célula , Línea Celular , Células Cultivadas , Reprogramación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Expresión Génica , Vectores Genéticos/genética , Humanos , Ratones , Neuronas/efectos de los fármacos , Retroviridae/genética , Transducción Genética , TransfecciónRESUMEN
Astrocytes are a viable source for generating new neurons via direct conversion. However, little is known about the neurogenic cascades triggered in astrocytes from different regions of the CNS. Here, we examine the transcriptome induced by the proneural factors Ascl1 and Neurog2 in spinal cord-derived astrocytes in vitro. Each factor initially elicits different neurogenic programs that later converge to a V2 interneuron-like state. Intriguingly, patch sequencing (patch-seq) shows no overall correlation between functional properties and the transcriptome of the heterogenous induced neurons, except for K-channels. For example, some neurons with fully mature electrophysiological properties still express astrocyte genes, thus calling for careful molecular and functional analysis. Comparing the transcriptomes of spinal cord- and cerebral-cortex-derived astrocytes reveals profound differences, including developmental patterning cues maintained in vitro. These relate to the distinct neuronal identity elicited by Ascl1 and Neurog2 reflecting their developmental functions in subtype specification of the respective CNS region.
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Astrocitos/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Reprogramación Celular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Médula Espinal/citología , Animales , Astrocitos/metabolismo , Biomarcadores/metabolismo , Fenómenos Electrofisiológicos , Ratones Endogámicos C57BL , Neuronas/metabolismo , Especificidad de Órganos , Transcripción GenéticaRESUMEN
During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression in mice. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9-mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor-binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act either redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.
Asunto(s)
Sistema de Transporte de Aminoácidos y+/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Organogénesis/genética , Factores de Transcripción Otx/genética , Retina/metabolismo , Animales , Secuencia de Bases , Elementos E-Box , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Noqueados , Ratones Transgénicos , Motivos de Nucleótidos , Factores de Transcripción Otx/metabolismo , Retina/embriologíaRESUMEN
Historically, the mammalian brain was thought to lack stem cells as no new neurons were found to be made in adulthood. That dogma changed â¼25 years ago with the identification of neural stem cells (NSCs) in the adult rodent forebrain. However, unlike rapidly self-renewing mature tissues (e.g., blood, intestinal crypts, skin), the majority of adult NSCs are quiescent, and those that become 'activated' are restricted to a few neurogenic zones that repopulate specific brain regions. Conversely, embryonic NSCs are actively proliferating and neurogenic. Investigations into the molecular control of the quiescence-to-proliferation-to-differentiation continuum in the embryonic and adult brain have identified proneural genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs) as critical regulators. These bHLH TFs initiate genetic programs that remove NSCs from quiescence and drive daughter neural progenitor cells (NPCs) to differentiate into specific neural cell subtypes, thereby contributing to the enormous cellular diversity of the adult brain. However, new insights have revealed that proneural gene activities are context-dependent and tightly regulated. Here we review how proneural bHLH TFs are regulated, with a focus on the murine cerebral cortex, drawing parallels where appropriate to other organisms and neural tissues. We discuss upstream regulatory events, post-translational modifications (phosphorylation, ubiquitinylation), protein-protein interactions, epigenetic and metabolic mechanisms that govern bHLH TF expression, stability, localization, and consequent transactivation of downstream target genes. These tight regulatory controls help to explain paradoxical findings of changes to bHLH activity in different cellular contexts.
RESUMEN
Heterogeneity of Purkinje cells (PCs) that are arranged into discrete longitudinally-striped compartments in the cerebellar cortex is related to the timing of PC generation. To understand the cerebellar compartmental organization, we mapped the PC birthdate (or differentiation timing) in the entire cerebellar cortex. We used the birthdate-tagging system of Neurog2-CreER (G2A) mice hybridized with the AldocV strain which visualizes the zebrin (aldolase C) longitudinal striped pattern. The birthdate-specific distribution pattern of PCs was arranged into longitudinally-oriented stripes consistently throughout almost all lobules except for the nodulus, paraflocculus, and flocculus, in which distinct stripes were observed. Boundaries of the birthdate stripes coincided with the boundary of zebrin stripes or located in the middle of a zebrin stripe. Each birthdate stripe contained PCs born in a particular period between embryonic day (E) 10.0 and E 13.5. In the vermis, PCs were chronologically distributed from lateral to medial stripes. In the paravermis, PCs of early birthdates were distributed in the long lateral zebrin-positive stripe (stripe 4+//5+) and the medially neighboring narrow zebrin-negative substripe (3d-//e2-), while PCs of late birthdates were distributed in the rest of all paravermal areas. In the hemisphere, PCs of early and late birthdates were intermingled in the majority of areas. The results indicate that the birthdate of a PC is a partial determinant for the zebrin compartment in which it is located. However, the correlation between the PC birthdate and the zebrin compartmentalization is complex and distinct among the vermis, paravermis, hemisphere, nodulus, and flocculus.
Asunto(s)
Vermis Cerebeloso , Células de Purkinje , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Corteza Cerebelosa/metabolismo , Cerebelo/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células de Purkinje/metabolismoRESUMEN
The mechanisms involved in regulation of quiescence, proliferation, and reprogramming of Neural Stem Progenitor Cells (NSPCs) of the mammalian brain are still poorly defined. Here, we studied the role of the transcriptional co-factor TAZ, regulated by the WNT and Hippo pathways, in the homeostasis of NSPCs. We found that, in the murine neurogenic niches of the striatal subventricular zone and the dentate gyrus granular zone, TAZ is highly expressed in NSPCs and declines with ageing. Moreover, TAZ expression is lost in immature neurons of both neurogenic regions. To characterize mechanistically the role of TAZ in neuronal differentiation, we used the midbrain-derived NSPC line ReNcell VM to replicate in a non-animal model the factors influencing NSPC differentiation to the neuronal lineage. TAZ knock-down and forced expression in NSPCs led to increased and reduced neuronal differentiation, respectively. TEADs-knockdown indicated that these TAZ co-partners are required for the suppression of NSPCs commitment to neuronal differentiation. Genetic manipulation of the TAZ/TEAD system showed its participation in transcriptional repression of SOX2 and the proneuronal genes ASCL1, NEUROG2, and NEUROD1, leading to impediment of neurogenesis. TAZ is usually considered a transcriptional co-activator promoting stem cell proliferation, but our study indicates an additional function as a repressor of neuronal differentiation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células-Madre Neurales/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Humanos , Ratones , NeurogénesisRESUMEN
The tuberal hypothalamus is comprised of the dorsomedial, ventromedial, and arcuate nuclei, as well as parts of the lateral hypothalamic area, and it governs a wide range of physiologies. During neurogenesis, tuberal hypothalamic neurons are thought to be born in a dorsal-to-ventral and outside-in pattern, although the accuracy of this description has been questioned over the years. Moreover, the intrinsic factors that control the timing of neurogenesis in this region are poorly characterized. Proneural genes, including Achate-scute-like 1 (Ascl1) and Neurogenin 3 (Neurog3) are widely expressed in hypothalamic progenitors and contribute to lineage commitment and subtype-specific neuronal identifies, but the potential role of Neurogenin 2 (Neurog2) remains unexplored. Birthdating in male and female mice showed that tuberal hypothalamic neurogenesis begins as early as E9.5 in the lateral hypothalamic and arcuate and rapidly expands to dorsomedial and ventromedial neurons by E10.5, peaking throughout the region by E11.5. We confirmed an outside-in trend, except for neurons born at E9.5, and uncovered a rostrocaudal progression but did not confirm a dorsal-ventral patterning to tuberal hypothalamic neuronal birth. In the absence of Neurog2, neurogenesis stalls, with a significant reduction in early-born BrdU+ cells but no change at later time points. Further, the loss of Ascl1 yielded a similar delay in neuronal birth, suggesting that Ascl1 cannot rescue the loss of Neurog2 and that these proneural genes act independently in the tuberal hypothalamus. Together, our findings show that Neurog2 functions as a classical proneural gene to regulate the temporal progression of tuberal hypothalamic neurogenesis.SIGNIFICANCE STATEMENT Here, we investigated the general timing and pattern of neurogenesis within the tuberal hypothalamus. Our results confirmed an outside-in trend of neurogenesis and uncovered a rostrocaudal progression. We also showed that Neurog2 acts as a classical proneural gene and is responsible for regulating the birth of early-born neurons within the ventromedial hypothalamus, acting independently of Ascl1 In addition, we revealed a role for Neurog2 in cell fate specification and differentiation of ventromedial -specific neurons. Last, Neurog2 does not have cross-inhibitory effects on Neurog1, Neurog3, and Ascl1 These findings are the first to reveal a role for Neurog2 in hypothalamic development.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Hipotálamo Medio/citología , Hipotálamo Medio/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Neurogénesis/fisiología , Neuronas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Femenino , Hipotálamo Medio/embriología , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , EmbarazoRESUMEN
The transcription factor Sox10 is a key regulator in the fate determination of a subpopulation of multipotent trunk neural crest (NC) progenitors toward glial cells instead of sensory neurons in the dorsal root ganglia (DRG). However, the mechanism by which Sox10 regulates glial cell fate commitment during lineage segregation remains poorly understood. In our study, we showed that the neurogenic determinant Neurogenin 2 (Neurog2) exhibited transient overlapping expression with Sox10 in avian trunk NC progenitors, which progressively underwent lineage segregation during migration toward the forming DRG. Gain- and loss-of-function studies revealed that the temporary expression of Neurog2 was due to Sox10 regulation of its protein stability. Transcriptional profiling identified Sox10-regulated F-box only protein (Fbxo9), which is an SCF (Skp1-Cul-F-box)-type ubiquitin ligase for Neurog2. Consistently, overexpression of Fbxo9 in NC progenitors down-regulated Neurog2 protein expression through ubiquitination and promoted the glial lineage at the expense of neuronal differentiation, whereas Fbxo9 knockdown resulted in the opposite phenomenon. Mechanistically, we found that Fbxo9 interacted with Neurog2 to promote its destabilization through the F-box motif. Finally, epistasis analysis further demonstrated that Fbxo9 and probably other F-box members mediated the role of Sox10 in destabilizing Neurog2 protein and directing the lineage of NC progenitors toward glial cells rather than sensory neurons. Altogether, these findings unravel a Sox10-Fbxo9 regulatory axis in promoting the glial fate of NC progenitors through Neurog2 destabilization.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas F-Box/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción SOXE/metabolismo , Raíces Nerviosas Espinales/metabolismo , Secuencias de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Embrión de Pollo , Proteínas F-Box/química , Proteínas F-Box/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Cresta Neural/citología , Cresta Neural/metabolismo , Neurogénesis , Unión Proteica , Estabilidad Proteica , Raíces Nerviosas Espinales/citologíaRESUMEN
Functionally distinct classes of dorsal root ganglia (DRG) somatosensory neurons arise from neural crest cells (NCCs) in two successive phases of differentiation assumed to be respectively and independently controlled by the proneural genes Neurog2 and Neurog1. However, the precise role of Neurog2 during this process remains unclear, notably because no neuronal loss has been reported hitherto in Neurog2-/- mutants. Here, we show that at trunk levels, Neurog2 deficiency impairs the production of subsets of all DRG neuron subtypes. We establish that this phenotype is highly dynamic and reflects multiple defects in NCC-derived progenitors, including somatosensory-to-melanocyte fate switch, apoptosis, and delayed differentiation which alters neuronal identity, all occurring during a narrow time window when Neurog2 temporarily controls onset of Neurog1 expression and neurogenesis. Collectively, these findings uncover a critical period of cell fate plasticity and vulnerability among somatosensory progenitors and establish that Neurog2 function in the developing DRG is broader than initially envisaged.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Plasticidad de la Célula/fisiología , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/metabolismo , Animales , Diferenciación Celular/fisiología , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiología , Regulación del Desarrollo de la Expresión Génica , Ratones , Neurogénesis/fisiología , Neuronas/metabolismo , Neuronas/fisiologíaRESUMEN
Neural progenitor cells (NPCs) are multipotent cells that have the potential to produce neurons and glial cells in the neural system. NPCs undergo identity maintenance or differentiation regulated by different kinds of transcription factors. Here we present evidence that ETV5, which is an ETS transcription factor, promotes the generation of glial cells and drives the neuronal subtype-specific genes in newly differentiated neurons from the human embryonic stem cells-derived NPCs. Next, we find a new role for ETV5 in the repression of NEUROG2 expression in NPCs. ETV5 represses NEUROG2 transcription via NEUROG2 promoter and requires the ETS domain. We identify ETV5 has the binding sites and is implicated in silent chromatin in NEUROG2 promoter by chromatin immunoprecipitation (ChIP) assays. Further, NEUROG2 transcription repression by ETV5 was shown to be dependent on a transcriptional corepressor (CoREST). During NPC differentiation toward neurons, ETV5 represses NEUROG2 expression and blocks the appearance of glutamatergic neurons. This finding suggests that ETV5 negatively regulates NEUROG2 expression and increases the number of GABAergic subtype neurons derived from NPCs. Thus, ETV5 represents a potent new candidate protein with benefits for the generation of GABAergic neurons.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Células-Madre Neurales/citología , Neurogénesis , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Autorrenovación de las Células , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Células Madre Embrionarias Humanas/metabolismo , Humanos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/genéticaRESUMEN
The adult mammalian brain contains an enormous variety of neuronal types, which are generally categorized in large groups, based on their neurochemical identity, hodological properties and molecular markers. This broad classification has allowed the correlation between individual neural progenitor populations and their neuronal progeny, thus contributing to probe the cellular and molecular mechanisms involved in neuronal identity determination during central nervous system (CNS) development. In this review, we discuss the contribution of the proneural genes Neurogenin2 (Neurog2) and Achaete-scute homolog 1 (Ascl1) for the specification of neuronal phenotypes in the developing neocortex, cerebellum and retina. Then, we revise recent data on astroglia cell lineage reprogramming into induced neurons using the same proneural proteins to compare the neuronal phenotypes obtained from astroglial cells originated in those CNS regions. We conclude that Ascl1 and Neurog2 have different contributions to determine neuronal fates, depending on the neural progenitor or astroglial population expressing those proneural factors. Finally, we discuss some possible explanations for these seemingly conflicting effects of Ascl1 and Neurog2 and propose future approaches to further dissect the molecular mechanisms of neuronal identity specification.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Astrocitos/metabolismo , Diferenciación Celular/genética , Linaje de la Célula/genética , Cerebelo , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Neocórtex , Proteínas del Tejido Nervioso/genética , Neurogénesis/fisiología , Neuronas/metabolismo , Retina , Células Madre/metabolismoRESUMEN
During embryonic retinal development, the bHLH factor Neurog2 regulates the temporal progression of neurogenesis, but no role has been assigned for this gene in the postnatal retina. Using Neurog2 conditional mutants, we found that Neurog2 is necessary for the development of an early, embryonic cohort of rod photoreceptors, but also required by both a subset of cone bipolar subtypes, and rod bipolars. Using transcriptomics, we identified a subset of downregulated genes in P2 Neurog2 mutants, which act during rod differentiation, outer segment morphogenesis or visual processing. We also uncovered defects in neuronal cell culling, which suggests that the rod and bipolar cell phenotypes may arise via more complex mechanisms rather than a simple cell fate shift. However, given an overall phenotypic resemblance between Neurog2 and Blimp1 mutants, we explored the relationship between these two factors. We found that Blimp1 is downregulated between E12-birth in Neurog2 mutants, which probably reflects a dependence on Neurog2 in embryonic progenitor cells. Overall, we conclude that the Neurog2 gene is expressed and active prior to birth, but also exerts an influence on postnatal retinal neuron differentiation.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Retina/citología , Neuronas Retinianas/metabolismo , Animales , Diferenciación Celular/fisiología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Neurogénesis/fisiología , Embarazo , Proteínas Represoras/genética , Retina/crecimiento & desarrollo , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/metabolismo , Neuronas Retinianas/citología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: In the developing mouse embryo, the bHLH transcription factor Neurog2 is transiently expressed by retinal progenitor cells and required for the initial wave of neurogenesis. Remarkably, another bHLH factor, Ascl1, normally not present in the embryonic Neurog2 retinal lineage, can rescue the temporal phenotypes of Neurog2 mutants. RESULTS: Here we show that Neurog2 simultaneously promotes terminal cell cycle exit and retinal ganglion cell differentiation, using mitotic window labeling and integrating these results with retinal marker quantifications. We also analyzed the transcriptomes of E12.5 GFP-expressing cells from Neurog2GFP/+ , Neurog2GFP/GFP , and Neurog2Ascl1KI/GFP eyes, and validated the most significantly affected genes using qPCR assays. CONCLUSIONS: Our data support the hypothesis that Neurog2 acts at the top of a retinal bHLH transcription factor hierarchy. The combined expression levels of these downstream factors are sufficiently induced by ectopic Ascl1 to restore RGC genesis, highlighting the robustness of this gene network during retinal ganglion cell neurogenesis. Developmental Dynamics 247:965-975, 2018. © 2018 Wiley Periodicals, Inc.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Ciclo Celular , Proteínas del Tejido Nervioso/fisiología , Neurogénesis , Células Ganglionares de la Retina/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/farmacología , Diferenciación Celular/efectos de los fármacos , Embrión de Mamíferos , Ratones , Transcriptoma/efectos de los fármacosRESUMEN
Proper embryonic development relies on a tight control of spatial and temporal gene expression profiles in a highly regulated manner. One good example is the ON/OFF switching of the transcription factor PAX6 that governs important steps of neurogenesis. In the neural tube PAX6 expression is initiated in neural progenitors through the positive action of retinoic acid signaling and downregulated in neuronal precursors by the bHLH transcription factor NEUROG2. How these two regulatory inputs are integrated at the molecular level to properly fine tune temporal PAX6 expression is not known. In this study we identified and characterized a 940-bp long distal cis-regulatory module (CRM), located far away from the PAX6 transcription unit and which conveys positive input from RA signaling pathway and indirect repressive signal(s) from NEUROG2. These opposing regulatory signals are integrated through HOMZ, a 94â¯bp core region within E940 which is evolutionarily conserved in distant organisms such as the zebrafish. We show that within HOMZ, NEUROG2 and RA exert their opposite temporal activities through a short 60â¯bp region containing a functional RA-responsive element (RARE). We propose a model in which retinoic acid receptors (RARs) and NEUROG2 repressive target(s) compete on the same DNA motif to fine tune temporal PAX6 expression during the course of spinal neurogenesis.
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Elementos de Facilitación Genéticos/genética , Tubo Neural/metabolismo , Neurogénesis/genética , Factor de Transcripción PAX6/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Ratones , Proteínas del Tejido Nervioso/metabolismo , Tubo Neural/embriología , Receptores de Ácido Retinoico/metabolismo , Transducción de Señal/fisiología , Pez CebraRESUMEN
Neurog2 is a crucial regulator of neuronal fate specification and differentiation in vivo and in vitro However, it remains unclear how Neurog2 transactivates neuronal genes that are silenced by repressive chromatin. Here, we provide evidence that the histone H3 lysine 9 demethylase KDM3A facilitates the Xenopus Neurog2 (formerly known as Xngnr1) chromatin accessibility during neuronal transcription. Loss-of-function analyses reveal that KDM3A is not required for the transition of naive ectoderm to neural progenitor cells but is essential for primary neuron formation. ChIP series followed by qPCR analyses reveal that Neurog2 promotes the removal of the repressive H3K9me2 marks and addition of active histone marks, including H3K27ac and H3K4me3, at the NeuroD1 and Tubb2b promoters; this activity depends on the presence of KDM3A because Neurog2, via its C-terminal domain, interacts with KDM3A. Interestingly, KDM3A is dispensable for the neuronal transcription initiated by Ascl1, a proneural factor related to neurogenin in the bHLH family. In summary, our findings uncover a crucial role for histone H3K9 demethylation during Neurog2-mediated neuronal transcription and help in the understanding of the different activities of Neurog2 and Ascl1 in initiating neuronal development.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Ectodermo/metabolismo , Femenino , Lisina/química , Neurogénesis , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Activación Transcripcional , Xenopus laevisRESUMEN
Background: Disrupted-in-schizophrenia 1 (DISC1) regulates neurogenesis and is a genetic risk factor for major psychiatric disorders. However, how DISC1 dysfunction affects neurogenesis and cell cycle progression at the molecular level is still unknown. Here, we investigated the role of DISC1 in regulating proliferation, migration, cell cycle progression and apoptosis in mouse neural stem/progenitor cells (MNSPCs) in vitro. Methods: MNSPCs were isolated and cultured from mouse fetal hippocampi. Retroviral vectors or siRNAs were used to manipulate DISC1 expression in MNSPCs. Proliferation, migration, cell cycle progression and apoptosis of altered MNSPCs were analyzed in cell proliferation assays (MTS), transwell system and flow cytometry. A neurogenesis specific polymerase chain reaction (PCR) array was used to identify genes downstream of DISC1, and functional analysis was performed through transfection of expression plasmids and siRNAs. Results: Loss of DISC1 reduced proliferation and migration of MNSPCs, while an increase in DISC1 led to increased proliferation and migration. Meanwhile, an increase in the proportion of cells in G0/G1 phase was concomitant with reduced levels of DISC1, but significant changes were not observed in the number MNSPCs undergoing apoptosis. Paired box gene 5 (Pax5), sex determining region Y-box 2 (Sox2), delta-like1 (Dll1) and Neurogenin2 (Neurog2) emerged as candidate molecules downstream of DISC1, and rescue experiments demonstrated that increased or decreased expression of either molecule regulated proliferation and migration in DISC1-altered MNSPCs. Conclusion: These results suggest that Pax5, Sox2, Dll1 and Neurog2 mediate DISC1 activity in MNSPC proliferation and migration.
RESUMEN
Until recently folate receptor alpha (FRα) has only been considered as a folate transporter. However, a novel role of FRα as a transcription factor was reported by our lab. More recently our lab showed a novel pleiotropic role of FRα: (a) direct transcriptional activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. These observations beg a question: "Can a simple molecule such as folate be used to manipulate the production and/or differentiation of endogenous neural stem cells (NSCs), which may hold promise for future therapies?" Conditions such as spinal cord injury, motor neuron diseases, Alzheimer's disease and multiple sclerosis may benefit from increasing stem cell pool and promoting specific pathways of differentiation. On the flip-side, these NSCs may also contribute to some CNS tumors therefore promoting differentiation could prove more beneficial. FRα may hold promises for both since it has the potential to remodel chromatin in a context dependent manner. In this commentary we discuss our previous data and new questions arising in the context of the new role for FRα.
RESUMEN
The T-box containing Tbr2 gene encodes for a transcription factor essential for the specification of the intermediate neural progenitors (INPs) originating the excitatory neurons of the cerebral cortex. However, its overall mechanism of action, direct target genes and cofactors remain unknown. Herein, we carried out global gene expression profiling combined with genome-wide binding site identification to determine the molecular pathways regulated by TBR2 in INPs. This analysis led to the identification of novel protein-protein interactions that control multiple features of INPs including cell-type identity, morphology, proliferation and migration dynamics. In particular, NEUROG2 and JMJD3 were found to associate with TBR2 revealing unexplored TBR2-dependent mechanisms. These interactions can explain, at least in part, the role of this transcription factor in the implementation of the molecular program controlling developmental milestones during corticogenesis. These data identify TBR2 as a major determinant of the INP-specific traits by regulating both genetic and epigenetic pathways.