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1.
Nat Commun ; 7: 10866, 2016 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-26946992

RESUMEN

Throughout vertebrates, cerebrospinal fluid-contacting neurons (CSF-cNs) are ciliated cells surrounding the central canal in the ventral spinal cord. Their contribution to modulate locomotion remains undetermined. Recently, we have shown CSF-cNs modulate locomotion by directly projecting onto the locomotor central pattern generators (CPGs), but the sensory modality these cells convey to spinal circuits and their relevance to innate locomotion remain elusive. Here, we demonstrate in vivo that CSF-cNs form an intraspinal mechanosensory organ that detects spinal bending. By performing calcium imaging in moving animals, we show that CSF-cNs respond to both passive and active bending of the spinal cord. In mutants for the channel Pkd2l1, CSF-cNs lose their response to bending and animals show a selective reduction of tail beat frequency, confirming the central role of this feedback loop for optimizing locomotion. Altogether, our study reveals that CSF-cNs constitute a mechanosensory organ operating during locomotion to modulate spinal CPGs.


Asunto(s)
Líquido Cefalorraquídeo/citología , Neuronas/citología , Médula Espinal/citología , Animales , Fenómenos Biomecánicos , Movimiento Celular , Líquido Cefalorraquídeo/metabolismo , Femenino , Masculino , Mecanorreceptores/citología , Mecanorreceptores/metabolismo , Neuronas/metabolismo , Médula Espinal/química , Médula Espinal/metabolismo , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
2.
Methods Cell Biol ; 104: 23-49, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21924155

RESUMEN

The Tol2 transposable element was originally found in the genome of the Japanese medaka fish (Oryzias latipes). Tol2 contains a gene encoding an active transposase that can catalyze DNA transposition in vertebrate cells. In zebrafish, Tol2 generates genomic integrations in the germ cells very efficiently. By using the Tol2 transposition system, we have developed important genetic methods including transgenesis, gene trapping, enhancer trapping, and the Gal4-UAS system in zebrafish. In this chapter, we describe how these methods can be performed.


Asunto(s)
Clonación Molecular/métodos , Elementos Transponibles de ADN , Mutagénesis Insercional/métodos , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Southern Blotting/métodos , Cromosomas Artificiales Bacterianos , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Microinyecciones/métodos , Recombinación Genética , Transfección/métodos
3.
BMC Dev Biol ; 10: 105, 2010 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-20950494

RESUMEN

BACKGROUND: We have developed genetic methods in zebrafish by using the Tol2 transposable element; namely, transgenesis, gene trapping, enhancer trapping and the Gal4FF-UAS system. Gene trap constructs contain a splice acceptor and the GFP or Gal4FF (a modified version of the yeast Gal4 transcription activator) gene, and enhancer trap constructs contain the zebrafish hsp70l promoter and the GFP or Gal4FF gene. By performing genetic screens using these constructs, we have generated transgenic zebrafish that express GFP and Gal4FF in specific cells, tissues and organs. Gal4FF expression is visualized by creating double transgenic fish carrying a Gal4FF transgene and the GFP reporter gene placed downstream of the Gal4-recognition sequence (UAS). Further, the Gal4FF-expressing cells can be manipulated by mating with UAS effector fish. For instance, when fish expressing Gal4FF in specific neurons are crossed with the UAS:TeTxLC fish carrying the tetanus neurotoxin gene downstream of UAS, the neuronal activities are inhibited in the double transgenic fish. Thus, these transgenic fish are useful to study developmental biology and neurobiology. DESCRIPTION: To increase the usefulness of the transgenic fish resource, we developed a web-based database named zTrap http://kawakami.lab.nig.ac.jp/ztrap/. The zTrap database contains images of GFP and Gal4FF expression patterns, and genomic DNA sequences surrounding the integration sites of the gene trap and enhancer trap constructs. The integration sites are mapped onto the Ensembl zebrafish genome by in-house Blat analysis and can be viewed on the zTrap and Ensembl genome browsers. Furthermore, zTrap is equipped with the functionality to search these data for expression patterns and genomic loci of interest. zTrap contains the information about transgenic fish including UAS reporter and effector fish. CONCLUSION: zTrap is a useful resource to find gene trap and enhancer trap fish lines that express GFP and Gal4FF in desired patterns, and to find insertions of the gene trap and enhancer trap constructs that are located within or near genes of interest. These transgenic fish can be utilized to observe specific cell types during embryogenesis, to manipulate their functions, and to discover novel genes and cis-regulatory elements. Therefore, zTrap should facilitate studies on genomics, developmental biology and neurobiology utilizing the transgenic zebrafish resource.


Asunto(s)
Animales Modificados Genéticamente/genética , Bases de Datos Genéticas , Elementos de Facilitación Genéticos , Pez Cebra/genética , Animales , Elementos Transponibles de ADN/genética , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Programas Informáticos , Transgenes , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo
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