Language：English   Publishing type：Research paper (scientific journal)  

Juvenile brain has a unique time window, or critical period, in which neuronal circuits are remodeled by experience. Mounting evidence indicates the importance of neuronal circuit rewiring in various neurodevelopmental disorders of human cognition. We previously showed that Otx2 homeoprotein, essential for brain formation, is recaptured during postnatal maturation of parvalbumin-positive interneurons (PV cells) to activate the critical period in mouse visual cortex. Cortical Otx2 is the only interneuron-enriched transcription factor known to regulate the critical period, but its downstream targets remain unknown. Here, we used ChIP-seq (chromatin immunoprecipitation sequencing) to identify genome-wide binding sites of Otx2 in juvenile mouse cortex, and interneuron-specific RNA-seq to explore the Otx2-dependent transcriptome. Otx2-bound genes were associated with human diseases such as schizophrenia as well as critical periods. Of these genes, expression of neuronal factors involved in transcription, signal transduction and mitochondrial function was moderately and broadly affected in Otx2-deficient interneurons. In contrast to reported binding sites in the embryo, genes encoding potassium ion transporters such as KV3.1 had juvenile cortex-specific binding sites, suggesting that Otx2 is involved in regulating fast-spiking properties during PV cell maturation. Moreover, transcripts of oxidative resistance-1 (Oxr1), whose promoter has Otx2 binding sites, were markedly downregulated in Otx2-deficient interneurons. Therefore, an important role of Otx2 may be to protect the cells from the increased oxidative stress in fast-spiking PV cells. Our results suggest that coordinated expression of Otx2 targets promotes PV cell maturation and maintains its function in neuronal plasticity and disease.

DOI： 10.3389/fnins.2017.00307

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2013.04.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

The calyx-type synapse of chick ciliary ganglion (CG) has been intensively studied for decades as a model system for the synaptic development, morphology and physiology. Despite recent advances in optogenetics probing and/or manipulation of the elementary steps of the transmitter release such as membrane depolarization and Ca2+ elevation, the current gene-manipulating methods are not suitable for targeting specifically the calyx-type presynaptic terminals. Here, we evaluated a method for manipulating the molecular and functional organization of the presynaptic terminals of this model synapse. We transfected progenitors of the Edinger-Westphal (EW) nucleus neurons with an EGFP expression vector by in ovo electroporation at embryonic day 2 (E2) and examined the CG at E8-14. We found that dozens of the calyx-type presynaptic terminals and axons were selectively labeled with EGFP fluorescence. When a Brainbow construct containing the membrane-tethered fluorescent proteins m-CFP, m-YFP and m-RFP, was introduced together with a Cre expression construct, the color coding of each presynaptic axon facilitated discrimination among inter-tangled projections, particularly during the developmental re-organization period of synaptic connections. With the simultaneous expression of one of the chimeric variants of channelrhodopsins, channelrhodopsin-fast receiver (ChRFR), and R-GECO1, a red-shifted fluorescent Ca2+-sensor, the Ca2+ elevation was optically measured under direct photostimulation of the presynaptic terminal. Although this optically evoked Ca2+ elevation was mostly dependent on the action potential, a significant component remained even in the absence of extracellular Ca2+. It is suggested that the photo-activation of ChRFR facilitated the release of Ca2+ from intracellular Ca2+ stores directly or indirectly. The above system, by facilitating the molecular study of the calyx-type presynaptic terminal, would provide an experimental platform for unveiling the molecular mechanisms underlying the morphology, physiology and development of synapses.

DOI： 10.1371/journal.pone.0059179

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

In vivo electroporation has served as an effective tool for the study of developmental biology. Here we report tetracycline inducible gene knockdown by electroporation. Our system consists of genome integration of a cassette encoding long double strand RNA (dsRNA) of a gene of interest by electroporation, transcription of which is assured by RNA polymerase II, and induction of transcription of dsRNA by tetracyclin. Long dsRNA decapped by ribozyme in the cassette and without poly A tail is processed into siRNA within nuclei. We could successfully induce knockdown of En2 and Coactosin by Dox administration.

DOI： 10.1111/j.1440-169X.2010.01229.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Coactosin is a 17 kDa actin binding protein that belongs to the actin depolymerizing factor/cofilin homology family. Coactosin inhibits barbed-end capping of actin filament, and is involved in actin polymerization. Coactosin is expressed in cephalic and trunk neural crest cells, cranial ganglia and dorsal root ganglia. Coactosin is also expressed in the cells that are forming mesonephric duct, and endodermal cells. Immunocytochemistry with anti-Coactosin antibody shows that Coactosin is localized in the cytoplasm, and associated with actin stress fibers in cultured neural crest cells. Coactosin is also expressed in the axon of oculomotor nerve and trigeminal nerve. In the growth cone of the oculomotor nerve axons, both Coactosin mRNA and protein were localized, which is indicative of the role of Coactosin in pathfinding of the growth cone. Coactosin is expressed in those that require dynamic and highly coordinated regulation of actin cytoskeleton, that is, neural crest cells, cells in the tip of the mesonephros, endodermal cells and axons.

DOI： 10.1111/j.1440-169X.2009.01146.x

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

In the field of developmental biology, electroporation has become a routine technique for gene transfer because of its simplicity and wide range of application. In chick embryos, the in ovo electroporation technique has enabled molecular analysis of developmental mechanisms in chick embryos and has revived chick embryos as the model animals in developmental biology (Muramatsu et al., 1997
Sakamoto et al., 1998
Funahashi et al., 1999
Nakamura et al., 2000). Especially, many gene analyses were conducted in developing neural tube, because the neural tube is a convenient tissue for application of in ovo electroporation. Here, we show the application of in ovo electroporation to the developmental neural tube in chick embryo. electroporation, only the anode side is transfected and the cathode side could be used as the control (Funahashi et al., 1999
Nakamura et al., 2000
Odani et al., 2008). © 2009 Springer Japan.

DOI： 10.1007/978-4-431-09427-2_2

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ELSEVIER IRELAND LTD  

DOI： 10.1016/j.neures.2010.07.1035

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