RESUMEN
The transcription factors neuronal helix-loop-helix protein (NEX)/mammalian atonal homolog 2 (Math-2), BETA2/neuronal determination factor (NeuroD), and NeuroD-related factor (NDRF)/NeuroD2 comprise a family of Drosophila atonal-related basic helix-loop-helix (bHLH) proteins with highly overlapping expression in the developing forebrain. The ability of BETA2/NeuroD and NDRF to convert ectodermal cells into neurons after mRNA injection into Xenopus oocytes suggested a role in specifying neuronal cell fate. However, neuronal bHLH genes are largely transcribed in CNS neurons, which are fully committed. Here we analyze a defect in mice lacking BETA2/NeuroD, and in NEX*BETA2/NeuroD double mutants, demonstrating that bHLH proteins are required in vivo for terminal neuronal differentiation. Most strikingly, presumptive granule cells of the dentate gyrus are generated but fail to mature, lack normal sodium currents, and show little dendritic arborization. Long-term hippocampal slice cultures demonstrate secondary alterations of entorhinal and commissural/associational projections. The primary developmental arrest appears to be restricted to granule cells in which an autoregulatory system involving all three neuronal bHLH genes has failed.
Asunto(s)
Giro Dentado/citología , Secuencias Hélice-Asa-Hélice/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Proteínas Virales , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Apoptosis/fisiología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Moléculas de Adhesión Celular Neuronal/análisis , Diferenciación Celular/fisiología , Giro Dentado/crecimiento & desarrollo , Proteínas de la Matriz Extracelular/análisis , Regulación del Desarrollo de la Expresión Génica , Etiquetado Corte-Fin in Situ , Integrasas/metabolismo , Antígeno Ki-67/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/química , Neuronas/enzimología , Técnicas de Placa-Clamp , Proteína Reelina , Serina Endopeptidasas , Activación Transcripcional/fisiologíaRESUMEN
Basic helix-loop-helix (bHLH) genes have emerged as important regulators of neuronal determination and differentiation in vertebrates. Three putative neuronal differentiation factors [NEX for neuronal helix-loop-helix protein-1 (mammalian atonal homolog-2), neuroD (beta-2), and NDRF for neuroD-related factor (neuroD2)] are highly homologous to each other in the bHLH region and comprise a new bHLH subfamily. To study the role of NEX, the first bHLH protein identified in this group, we have disrupted the NEX gene by homologous recombination. NEX-deficient mice have no obvious developmental defect, and CNS neurons appear fully differentiated. To investigate further whether the absence of NEX is compensated for by neuroD and NDRF, we compared the spatiotemporal expression of all three genes. We demonstrate, by in situ hybridization, that the transcription patterns of NEX, neuroD, and NDRF genes are highly overlapping in the developing CNS of normal rats between embryonic day 12 and adult stages but are not strictly identical. The most prominent transcription of each gene marks the dorsal neuroepithelium of the telencephalon in early development and is sustained in the adult neocortex, hippocampus, and cerebellum. In general, neuroD provides the earliest marker of neuronal differentiation in any given region compared with NDRF or NEX. Whereas a few CNS regions are specific for neuroD, no region was detected in which solely NEX or NDRF is expressed. This suggests that the function of the mutant NEX gene in neuronal differentiation is compensated for by neuroD and NDRF and that, in analogy with myogenic bHLH proteins, neuronal differentiation factors are at least in part equivalent in function.
Asunto(s)
Ratones Transgénicos/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo/metabolismo , Diferenciación Celular/fisiología , Desarrollo Embrionario y Fetal/fisiología , Expresión Génica/fisiología , Secuencias Hélice-Asa-Hélice/genética , Ratones , Neuronas/citología , Neuropéptidos/genética , Ratas , Ratas Sprague-DawleyRESUMEN
The neuropeptide head activator (HA) acts as an autocrine growth factor for the neural cell line NH15-CA2. Cell proliferation is increased in the presence of HA and inhibited by HA peptide-specific antisera. Stimulation of cellular proliferation is visible 2 h after HA application as an increase in cells in mitosis. HA has no direct effect on stimulating DNA synthesis. HA thus functions as a control signal in the G2/mitosis transition and not in the G1/S transition. Receptors for HA are present on small round cells in clusters of foci and not on cells with differentiated morphology, suggesting cell-cycle-dependent HA receptor expression.