RESUMEN
Precise control of neuronal migration is essential for proper function of the brain. Taking a forward genetic screen, we isolated a mutant mouse with defects in interneuron migration. By genetic mapping, we identified a frame shift mutation in the pericentrin (Pcnt) gene. The Pcnt gene encodes a large centrosomal coiled-coil protein that has been implicated in schizophrenia. Recently, frame shift and premature termination mutations in the pericentrin (PCNT) gene were identified in individuals with Seckel syndrome and microcephalic osteodysplastic primordial dwarfism (MOPD II), both of which are characterized by greatly reduced body and brain sizes. The mouse Pcnt mutant shares features with the human syndromes in its overall growth retardation and reduced brain size. We found that dorsal lateral ganglionic eminence (dLGE)-derived olfactory bulb interneurons are severely affected and distributed abnormally in the rostral forebrain in the mutant. Furthermore, mutant interneurons exhibit abnormal migration behavior and RNA interference knockdown of Pcnt impairs cell migration along the rostal migratory stream (RMS) into the olfactory bulb. These findings indicate that pericentrin is required for proper migration of olfactory bulb interneurons and provide a developmental basis for association of pericentrin function with interneuron defects in human schizophrenia.
Asunto(s)
Antígenos/genética , Movimiento Celular/fisiología , Interneuronas/citología , Mutación , Bulbo Olfatorio/metabolismo , Animales , Centrosoma/metabolismo , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
The Hedgehog (Hh) signaling pathway regulates development in animals ranging from flies to humans. Although its framework is conserved, differences in pathway components have been reported. A kinesin-like protein, Costal2 (Cos2), plays a central role in the Hh pathway in flies. Knockdown of a zebrafish homolog of Cos2, Kif7, results in ectopic Hh signaling, suggesting that Kif7 acts primarily as a negative regulator of Hh signal transduction. However, in vitro analysis of the function of mammalian Kif7 and the closely related Kif27 has led to the conclusion that neither protein has a role in Hh signaling. Using Kif7 knockout mice, we demonstrate that mouse Kif7, like its zebrafish and Drosophila homologs, plays a role in transducing the Hh signal. We show that Kif7 accumulates at the distal tip of the primary cilia in a Hh-dependent manner. We also demonstrate a requirement for Kif7 in the efficient localization of Gli3 to cilia in response to Hh and for the processing of Gli3 to its repressor form. These results suggest a role for Kif7 in coordinating Hh signal transduction at the tip of cilia and preventing Gli3 cleavage into a repressor form in the presence of Hh.
Asunto(s)
Cilios/metabolismo , Desarrollo Embrionario/fisiología , Proteínas Hedgehog/metabolismo , Cinesinas/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Células Cultivadas , Cartilla de ADN/genética , Genotipo , Proteínas Fluorescentes Verdes , Inmunoprecipitación , Cinesinas/fisiología , Ratones , Ratones Noqueados , Proteína Gli3 con Dedos de ZincRESUMEN
In the alkane-assimilating yeast Yarrowia lipolytica, the expression of ALK1, a gene encoding cytochrome P450 that catalyzes the first step of n-alkane oxidation, is induced by n-alkanes. We previously demonstrated that two basic helix-loop-helix proteins, Yas1p and Yas2p, activate the transcription of ALK1 in an alkane-dependent manner by forming a heterocomplex and binding to alkane-responsive element 1 (ARE1), a cis-acting element in the ALK1 promoter. Here we identified an Opi1 family transcription factor, Yas3p, involved in the alkane-dependent transcription regulation of ALK genes. Deletion of YAS3 caused a significant increase in ALK1 mRNA in cells grown on glucose, glycerol, and n-alkanes. The YAS3 deletion also resulted in a marked elevation of reporter gene expression driven by an ARE1-containing promoter on glycerol and n-decane. Bacterially expressed Yas3p bound specifically to Yas2p, but not to Yas1p, in vitro. In addition, although green fluorescent protein-tagged Yas3p was localized in the nucleus in glucose-containing medium, it changed its localization to an endoplasmic reticulum-like compartment upon transfer to medium containing n-decane. These findings suggest that Yas3p functions as a master regulator of transcriptional response, which changes its localization between the nucleus and endoplasmic reticulum membrane in response to different carbon sources. Furthermore, quantitative real time PCR analysis of 12 ALK genes in YAS1, YAS2, and YAS3 deletion mutants suggested that Yas3p is involved in the transcriptional repression of a variety of ALK genes, including ALK1. In contrast, YAS3 deletion did not affect the mRNA level of an INO1 ortholog in Y. lipolytica, indicating functional diversity of Opi1 family transcription factors.
Asunto(s)
Alcanos/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Proteínas Fúngicas/biosíntesis , Regulación Enzimológica de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Factores de Transcripción/metabolismo , Yarrowia/enzimología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Alcanos/farmacología , Secuencia de Bases , Núcleo Celular/enzimología , Núcleo Celular/genética , Sistema Enzimático del Citocromo P-450/genética , Citoplasma/enzimología , Citoplasma/genética , Proteínas Fúngicas/genética , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Oxidación-Reducción , ARN de Hongos/biosíntesis , ARN de Hongos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factores de Transcripción/genética , Yarrowia/genéticaRESUMEN
The expression of the ALK1 gene, which encodes cytochrome P450, catalyzing the first step of alkane oxidation in the alkane-assimilating yeast Yarrowia lipolytica, is highly regulated and can be induced by alkanes. Previously, we identified a cis-acting element (alkane-responsive element 1 [ARE1]) in the ALK1 promoter. We showed that a basic helix-loop-helix (bHLH) protein, Yas1p, binds to ARE1 in vivo and mediates alkane-dependent transcription induction. Yas1p, however, does not bind to ARE1 by itself in vitro, suggesting that Yas1p requires another bHLH protein partner for its DNA binding, as many bHLH transcription factors function by forming heterodimers. To identify such a binding partner of Yas1p, here we screened open reading frames encoding proteins with the bHLH motif from the Y. lipolytica genome database and identified the YAS2 gene. The deletion of the YAS2 gene abolished the alkane-responsive induction of ALK1 transcription and the growth of the yeast on alkanes. We revealed that Yas2p has transactivation activity. Furthermore, Yas1p and Yas2p formed a protein complex that was required for the binding of these proteins to ARE1. These findings allow us to postulate a model in which bHLH transcription factors Yas1p and Yas2p form a heterocomplex and mediate the transcription induction in response to alkanes.