RESUMEN

Hyperactivated mTOR signaling in the developing brain has been implicated in multiple forms of pathology including tuberous sclerosis complex (TSC). To date, various phenotypic defects such as cortical lamination irregularity, subependymal nodule formation, dysmorphic astrocyte differentiation and dendritic malformation have been described for patients and animal models. However, downstream networks affected in the developing brain by hyperactivated mTOR signaling have yet to be characterized. Here, we present an integrated analysis of transcriptomes and proteomes generated from wild-type and Tsc1/Emx1-Cre forebrains. This led to comprehensive lists of genes and proteins whose expression levels were altered by hyperactivated mTOR signaling. Further incorporation of TSC patient data followed by functional enrichment and network analyses pointed to changes in molecular components and cellular processes associated with neuronal differentiation and morphogenesis as the key downstream events underlying developmental and morphological defects in TSC. Our results provide novel and fundamental molecular bases for understanding hyperactivated mTOR signaling-induced brain defects which can in turn facilitate identification of potential diagnostic markers and therapeutic targets for mTOR signaling-related neurological disorders.

Asunto(s)

Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Prosencéfalo/metabolismo , Proteoma , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma , Cromatografía Liquida , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Proteómica/métodos , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem

RESUMEN

How a pool of undifferentiated neural progenitor cells is maintained in the developing nervous system is an issue that remains unresolved. One of the key transcription factors for self-renewal of these cells is Sox2, the forced expression of which has been shown to inhibit neuronal differentiation in vivo. To dissect the molecular mechanisms of Sox2 activity, a ChIP-on-chip assay has been carried out for Sox2, and multiple candidate direct target genes have been isolated. In this report, we provide evidence indicating that Sox6, which like Sox2 belongs to the SRY-related HMG box transcription factor family, is a bona-fide direct regulatory target of Sox2. In vivo, Sox6 expression is seen with a temporal lag in Sox2-positive neural precursor cells in the ventricular zone, and Sox2 promotes expression of Sox6 as a transcriptional activator. Interestingly, gain- and loss-of-function assays indicate that Sox6 in turn is required for the maintenance of Sox2 expression, suggesting that a positive feedback loop, which functions to inhibit premature neuronal differentiation, exists between the two transcription factors.

Asunto(s)

Diferenciación Celular/fisiología , Sistema Nervioso Central/embriología , Retroalimentación Fisiológica/fisiología , Neurogénesis/fisiología , Neuronas/fisiología , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción SOXD/metabolismo , Animales , Secuencia de Bases , Embrión de Pollo , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXD/genética , Análisis de Secuencia de ADN

RESUMEN

We report isolation of novel splice variants of chicken Neuronal Per-Arnt-Sim domain protein 3 (cNPAS3) gene distinct from the previously predicted cNPAS3 at the 5' end. Newly identified cNPAS3 splice variants feature N-terminus coding sequences with high degrees of homology to human NPAS3 (hNAPS3). We also show that the alternative splicing pattern of NPAS3 is conserved between chicken and human. RNA in situ hybridization indicated that the expression of cNPAS3 in the developing central nervous system (CNS) is limited to the ventricular zone and only partially overlaps with that of chicken Reelin (cReelin), the only known regulatory target gene of NPAS3 in the adult brain. Overexpression of cNPAS3 by in ovo electroporation had little effect on the expression of Sox2, a marker for neural precursors, or of Isl1/2, a marker for early differentiating motor neurons. Taken together with the little effect of cNPAS3 overexpression on cReelin, it is noted that the function of NPAS3 in the developing CNS remains to be determined. Still, identification of proper cDNA sequences for cNPAS3 should represent a solid beginning of the understanding process.

Asunto(s)

Empalme Alternativo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Sistema Nervioso Central/metabolismo , Pollos/genética , Regulación del Desarrollo de la Expresión Génica , Neuronas Motoras/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Pollos/crecimiento & desarrollo , Pollos/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Secuencias Hélice-Asa-Hélice , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Datos de Secuencia Molecular , Neuronas Motoras/citología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Estructura Terciaria de Proteína , Proteína Reelina , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Here we describe characterization of chicken neuronal Per-Arnt-Sim domain 3 (NPAS3) gene during embryogenesis including examinations of expression pattern and function of the gene. RTPCR assay showed that the primary tissue of expression for this gene is the central nervous system (CNS) while RNA in situ hybridization assay confirmed that NPAS3 was expressed in the ventricular zone of developing neural tube as early as Hamburger-Hamilton (HH) stage 20. Ectopic over-expression of the gene in ovo in the developing chicken neural tube by electroporation had little effect on stem cell population, overall neurogenesis, and motor neuron differentiation. We discuss the implications of our observation.