RESUMEN
Tlx3 (HOX11L2) is regarded as one of the selector genes in excitatory versus inhibitory fate specification of neurons in distinct regions of the nervous system. Expression of Tlx3 in a post-mitotic immature neuron favors a glutamatergic over GABAergic fate. The factors that regulate Tlx3 have immense importance in the fate specification of glutamatergic neurons. Here, we have shown that Notch target gene, Hes-1, negatively regulates Tlx3 expression, resulting in decreased generation of glutamatergic neurons. Down-regulation of Hes-1 removed the inhibition on Tlx3 promoter, thus promoting glutamatergic differentiation. Promoter-protein interaction studies with truncated/mutated Hes-1 protein suggested that the co-repressor recruitment mediated through WRPW domain of Hes-1 has contributed to the repressive effect. Our results clearly demonstrate a new and unique role for canonical Notch signaling through Hes-1, in neurotransmitter/subtype fate specification of neurons in addition to its known functional role in proliferation/maintenance of neural progenitors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Células-Madre Neurales/citología , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Sitios de Unión , Diferenciación Celular , Línea Celular , ADN/metabolismo , Células Madre Embrionarias/citología , Ácido Glutámico/farmacología , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Ratones , Datos de Secuencia Molecular , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Factor de Transcripción HES-1 , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Hes-1 and Hes-5 are downstream effectors of Notch signaling that are known to be involved in different aspects of neural stem cell proliferation and differentiation. Evidence has emerged that Hes-1 expression can be regulated by alternate signaling pathways independent of canonical Notch/CBF1 interaction. This context-dependent differential regulation of Hes-1 expression in neural progenitor gains a lot of importance as it would help in its exponential expansion without the requirement of interaction from neighboring cells during development. Here, we have clearly demonstrated the existence of a population of neural progenitors with Notch/CBF1-independent Hes-1 expression in vitro. Further analysis demonstrated the role of FGF2 in activating Hes-1 expression through the direct binding of ATF2, a JNK downstream target, on Hes-1 promoter. This raises the possibility for the existence of two distinct populations of neural progenitors - one maintained by Hes-1 expression exclusively through Notch-independent mechanism and the other mediating Hes-1 expression through both canonical Notch and FGF2-ATF2 pathway. This alternative pathway will insure a constant expression of Hes-1 even in the absence of canonical Notch intracellular domain-mediated signaling, thereby maintaining a pool of proliferating neural progenitors required during development.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sistema Nervioso Central/embriología , Proteínas de Homeodominio/metabolismo , Neuronas/metabolismo , Receptores Notch/metabolismo , Células Madre/metabolismo , Factor de Transcripción Activador 2/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/fisiología , Línea Celular , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Neuronas/citología , Regiones Promotoras Genéticas/genética , Receptores Notch/genética , Transducción de Señal/fisiología , Células Madre/citología , Factor de Transcripción HES-1 , Activación Transcripcional/fisiologíaRESUMEN
The role of Wnt5a has been extensively explored in various aspects of development but its role in cerebellar development remains elusive. Here, for the first time we unravel the expression pattern and functional significance of Wnt5a in cerebellar development using Wnt5a-/- and Nestin-Cre mediated conditional knockout mouse models. We demonstrate that loss of Wnt5a results in cerebellar hypoplasia and depletion of GABAergic and glutamatergic neurons. Besides, Purkinje cells of the mutants displayed stunted, poorly branched dendritic arbors. Furthermore, we show that the overall reduction is due to decreased radial glial and granule neuron progenitor cell proliferation. At molecular level we provide evidence for non-canonical mode of action of Wnt5a and its regulation over genes associated with progenitor proliferation. Altogether our findings imply that Wnt5a signaling is a crucial regulator of cerebellar development and would aid in better understanding of cerebellar disease pathogenesis caused due to deregulation of Wnt signaling.
Asunto(s)
Cerebelo/metabolismo , Neurogénesis/genética , Proteína Wnt-5a/genética , Animales , Biomarcadores , Proliferación Celular , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Neuronas GABAérgicas/metabolismo , Eliminación de Gen , Expresión Génica , Ratones , Ratones Noqueados , Ratones Transgénicos , Mutación , Células-Madre Neurales/metabolismo , Células de Purkinje/metabolismo , Proteína Wnt-5a/metabolismoRESUMEN
Homeobox gene Tlx3 is known to promote glutamatergic differentiation and is expressed in post-mitotic neurons of CNS. Contrary to this here, we discovered that Tlx3 is expressed in the proliferating progenitors of the external granule layer in the cerebellum, and examined factors that regulate this expression. Using Pax6(-/-)Sey mouse model and molecular interaction studies we demonstrate Pax6 is a key activator of Tlx3 specifically in cerebellum, and induces its expression starting at embryonic day (E)15. By Postnatal day (PN)7, Tlx3 is expressed in a highly restricted manner in the cerebellar granule neurons of the posterior cerebellar lobes, where it is required for the restricted expression of nicotinic cholinergic receptor-α3 subunit (Chrnα3) and other genes involved in formation of synaptic connections and neuronal migration. These results demonstrate a novel role for Tlx3 and indicate that Pax6-Tlx3 expression and interaction is part of a region specific regulatory network in cerebellum and its deregulation during development could possibly lead to Autistic spectral disorders (ASD).