RESUMEN
A quasisteady reversed shear plasma with a large bootstrap current fraction ( approximately 80%) has been obtained for the first time in the JT-60U tokamak. The shrinkage of reversed shear region was suppressed by the bootstrap current peaked at the internal transport barrier (ITB) layer and the ITBs at a large radius were sustained, which, by combination with an H-mode edge pedestal, resulted in a high confinement or 2.2 times the H-mode scaling for 6 times energy confinement time or 2.7 s. Furthermore, a full noninductive current drive was obtained by the bootstrap current and the beam driven current.
RESUMEN
Islet-2 is a LIM/homeodomain-type transcription factor of the Islet-1 family expressed in embryonic zebrafish. Two Islet-2 molecules bind to the LIM domain binding protein (Ldb) dimers. Overexpression of the LIM domains of Islet-2 or the LIM-interacting domain of Ldb proteins prevented binding of Islet-2 to Ldb proteins in vitro and caused similar in vivo defects in positioning, peripheral axonal outgrowth, and neurotransmitter expression by the Islet-2-positive primary sensory and motor neurons as the defects induced by injection of Islet-2-specific antisense morpholino oligonucleotide. These and other experiments, i.e., mosaic analysis, coexpression of full-length Islet-2, and overexpression of the chimeric LIM domains derived from two different Islet-1 family members, demonstrated that Islet-2 regulates neuronal differentiation by forming a complex with Ldb dimers and possibly with some other Islet-2-specific cofactors.
Asunto(s)
Axones/fisiología , Proteínas de Homeodominio/metabolismo , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas Aferentes/fisiología , Pez Cebra/embriología , Pez Cebra/genética , Animales , Sitios de Unión , Embrión no Mamífero/fisiología , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas con Homeodominio LIM , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Mosaicismo , Proteínas del Tejido Nervioso/genética , Oligodesoxirribonucleótidos Antisentido/farmacología , Biosíntesis de Proteínas , ARN Mensajero/genética , Factores de Transcripción , Transcripción GenéticaRESUMEN
We generated germ line-transmitting transgenic zebrafish that express green fluorescent protein (GFP) in the cranial motor neurons. This was accomplished by fusing GFP sequences to Islet-1 promoter/enhancer sequences that were sufficient for neural-specific expression. The expression of GFP by the motor neurons in the transgenic fish enabled visualization of the cell bodies, main axons, and the peripheral branches within the muscles. GFP-labeled motor neurons could be followed at high resolution for at least up to day four, when most larval neural circuits become functional, and larvae begin to swim and capture prey. Using this line, we analyzed axonal outgrowth by the cranial motor neurons. Furthermore, by selective application of DiI to specific GFP-positive nerve branches, we showed that the two clusters of trigeminal motor neurons in rhombomeres 2 and 3 innervate different peripheral targets. This finding suggests that the trigeminal motor neurons in the two clusters adopt distinct fates. In future experiments, this transgenic line of zebrafish will allow for a genetic analysis of cranial motor neuron development.
Asunto(s)
Proteínas de Homeodominio/genética , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso , Regiones Promotoras Genéticas/fisiología , Nervio Trigémino/citología , Animales , Carbocianinas , Elementos de Facilitación Genéticos/fisiología , Nervio Facial/citología , Nervio Facial/crecimiento & desarrollo , Colorantes Fluorescentes , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Nervio Glosofaríngeo/citología , Nervio Glosofaríngeo/crecimiento & desarrollo , Proteínas Fluorescentes Verdes , Proteínas con Homeodominio LIM , Larva/citología , Larva/fisiología , Músculo Esquelético/inervación , Mutagénesis/fisiología , Neuronas Aferentes/citología , Neuronas Aferentes/fisiología , Nervio Oculomotor/citología , Nervio Oculomotor/crecimiento & desarrollo , Organismos Modificados Genéticamente , Factores de Transcripción , Nervio Trigémino/crecimiento & desarrollo , Nervio Vago/citología , Nervio Vago/crecimiento & desarrollo , Pez CebraAsunto(s)
Tipificación del Cuerpo/genética , Proteínas del Tejido Nervioso , Nervio Trigémino/embriología , Pez Cebra/embriología , Pez Cebra/genética , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/genética , Encéfalo/embriología , Ectodermo/citología , Embrión no Mamífero , Proteínas de Homeodominio/genética , Proteínas con Homeodominio LIM , Neuronas Motoras/citología , Cresta Neural/citología , Neuronas Aferentes/fisiología , Factores de Transcripción , Nervio Trigémino/citologíaRESUMEN
Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento , Hipocampo/embriología , Moléculas de Adhesión de Célula Nerviosa/genética , Neuronas/fisiología , Péptidos , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Clonación Molecular , Drosophila , Proteínas de Drosophila , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/fisiología , Péptidos y Proteínas de Señalización Intercelular , Proteínas de la Membrana , Datos de Secuencia Molecular , Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/fisiología , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Secuencias Repetitivas de Aminoácido , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Pez CebraRESUMEN
F-spondin is a secreted protein expressed at high levels by the floor plate cells. The C-terminal half of the protein contains six thrombospondin type 1 repeats, while the N-terminal half exhibited virtually no similarity to any other protein until recently, when a Drosophila gene termed M-spondin was cloned; its product was found to share two conserved domains with the N-terminal half of F-spondin. We report the molecular cloning of four zebrafish genes encoding secreted proteins with these conserved domains. Two are zebrafish homologs of F-spondin, while the other two, termed mindin1 and mindin2, encode mutually related novel proteins, which are more related to the Drosophila M-spondin than to F-spondin. During embryonic development, all four genes are expressed in the floor plate cells. In addition to the floor plate, mindin1 is expressed in the hypochord cells, while mindin2 is expressed in the sclerotome cells. When ectopically expressed, Mindin proteins selectively accumulate in the basal lamina, suggesting that Mindins are extracellular matrix (ECM) proteins with high affinity to the basal lamina. We also report the spatial distribution of one of the F-spondin proteins, F-spondin2. F-spondin2 is localized to the thread-like structure in the central canal of the spinal cord, which is likely to correspond to Reissner's fiber known to be present in the vertebrate phylum. In summary, our study has defined a novel gene family of ECM molecules in the vertebrate, all of which may potentially be involved in development of the midline structure.
Asunto(s)
Proteínas de la Matriz Extracelular/fisiología , Regulación del Desarrollo de la Expresión Génica , Sustancias de Crecimiento , Moléculas de Adhesión de Célula Nerviosa/fisiología , Péptidos , Proteínas de Pez Cebra , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Consenso , Proteínas de Drosophila , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero/fisiología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Proteínas de la Membrana , Datos de Secuencia Molecular , Familia de Multigenes , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Despite a number of reports on transgenic zebrafish, there have been no reports on transgenic zebrafish in which the gene is under the control of a promoter of zebrafish origin. Neither have there been reports on transgenic zebrafish in which the gene is under the control of a tissue-specific promoter/enhancer. To investigate whether it is possible to generate transgenic zebrafish which reliably express a reporter gene in specific tissues, we have isolated a zebrafish muscle-specific actin (alpha-actin) promoter and generated transgenic zebrafish in which the green fluorescent protein (GFP) reporter gene was driven by this promoter. In total, 41 GFP-expressing transgenic lines were generated with a frequency of as high as 21% (41 of 194), and GFP was specifically expressed throughout muscle cells in virtually all of the lines (40 of 41). Nonexpressing transgenic lines were rare. This demonstrates that a tissue-specific promoter can reliably drive reporter gene expression in transgenic zebrafish in a manner identical to the control of the endogeneous expression of the gene. Levels of GFP expression varied greatly from line to line; i.e., fluorescence was very weak in some lines, while it was extremely high in others. We also isolated a zebrafish cytoskeletal beta-actin promoter and generated transgenic zebrafish using a beta-actin-GFP construct. In all of the four lines generated, GFP was expressed throughout the body like the beta-actin gene, demonstrating that consistent expression could also be achieved in this case. In the present study, we also examined the effects of factors which potentially affect the transgenic frequency or expression levels. The following results were obtained: (i) expression levels of GFP in the injected embryo were not strongly correlated to transgenic frequency; (ii) the effect of the NLS peptide (SV40 T antigen nuclear localization sequence), which has been suggested to facilitate the transfer of a transgene into embryonic nuclei, remained to be elusive; (iii) a plasmid vector sequence placed upstream of the construct might reduce the expression levels of the reporter gene.
Asunto(s)
Actinas/genética , Regulación del Desarrollo de la Expresión Génica , Músculo Esquelético/embriología , Regiones Promotoras Genéticas/genética , Pez Cebra/genética , Actinas/biosíntesis , Animales , Animales Modificados Genéticamente , Antígenos Transformadores de Poliomavirus/genética , Clonación Molecular , Embrión no Mamífero/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Músculo Esquelético/metabolismo , Especificidad de Órganos , Proteínas Recombinantes de Fusión/biosíntesis , Virus 40 de los Simios/genética , Transgenes , Pez Cebra/embriologíaRESUMEN
We isolated and characterized the eagle gene, encoding a member of the steroid receptor superfamily in Drosophila. In the central nervous system eagle RNA was expressed in a limited number of cells. During stages 10 and 11, eagle RNA expression was observed in four neuroblasts, NB2-4, NB3-3, NB6-4 and NB7-3. Except for NB6-4, eagle RNA expression reached a maximum at the very beginning of expression or in the period of neuroblast delamination. Weak eagle RNA expression was also observed in a few putative progeny of NB7-3 during stages, late 11 and 12. All eagle RNA in abdominal segments disappeared at stage 13. Using an eagle-kinesin-lacZ fusion gene as a reporter, the division, migration, and axonogenesis in eagle-positive cells and their derivatives were examined. At stage 14, several types of neural or glial cells were detected which include EG and EW interneurons joining to the anterior and posterior commissures, respectively. Lack of eagle expression caused altered axonogenesis in an appreciable fraction of eagle-Kinesin-LacZ-positive neurons. Some EG cells failed to acquire the neural fate or underwent an extremely delayed differentiation, while EW neurons produced neurites in abnormal directions, suggesting that eagle may play a critical role in development of the progeny of eagle-positive neuroblasts.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/embriología , Expresión Génica , Genes de Insecto , Familia de Multigenes , Sistema Nervioso/embriología , Neuropéptidos/biosíntesis , Receptores de Esteroides/biosíntesis , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Drosophila melanogaster/genética , Embrión no Mamífero/fisiología , Biblioteca de Genes , Cinesinas/biosíntesis , Datos de Secuencia Molecular , Mutagénesis Insercional , Sistema Nervioso/metabolismo , Neuronas/citología , Neuronas/fisiología , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo Restrictivo , beta-Galactosidasa/análisis , beta-Galactosidasa/biosíntesisRESUMEN
A Drosophila gene encoding a gamma-type isozyme of phosphoinositide-specific phospholipase C (PLC) was isolated and characterized. The gene, termed plc-gamma d, was mapped at position 14B-C of the X chromosome. The encoded protein, termed PLC-gamma D, contains X and Y regions, common to all known PLC isozymes. The two regions are split by a Z region that comprises two src homology 2 and one src homology 3 domains and is characteristic of gamma-type mammalian PLC (PLC-gamma 1 and -gamma 2). The deduced amino acid sequence of PLC-gamma D shows overall similarity to mammalian PLC-gamma s; no large deletion was observed except the short C-terminal extended region. In particular, the two split catalytic domains (X and Y regions) and the regulatory Z region including the src homology 2 and src homology 3 domains are well conserved. The mRNA is expressed throughout development, but expression is relatively higher during the embryonic stage, suggesting fundamental and important roles in both cell proliferation and differentiation. Distribution of the mRNA during embryogenesis, as analyzed by whole amount in situ hybridization, revealed that the mRNA emerges and reaches maximum levels at the cellular blastoderm stage and then decreases rapidly to a lower level. In later embryonic stages, invaginated anterior and posterior midgut primordia show high levels of mRNA expression, and fused midgut also maintains a high level of expression. In other tissues and cells, the mRNA was detected at lower levels. These results indicate that Drosophila PLC-gamma may be involved in universal cellular processes mediated possibly by receptor tyrosine kinases during embryogenesis and may also play specific roles during cellularization and midgut differentiation.
Asunto(s)
Drosophila/embriología , Drosophila/enzimología , Genes de Insecto/genética , Isoenzimas/genética , Fosfolipasas de Tipo C/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Blastodermo/enzimología , Mapeo Cromosómico , Cromosomas/ultraestructura , Clonación Molecular , Drosophila/genética , Drosophila/crecimiento & desarrollo , Endodermo/enzimología , Hibridación in Situ , Isoenzimas/biosíntesis , Datos de Secuencia Molecular , Fosfolipasa C gamma , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Fosfolipasas de Tipo C/biosíntesis , Cromosoma XRESUMEN
The complete nucleotide sequence of the coding region of hedgehog (hh), a segment-polarity gene in Drosophila melanogaster, was determined. The gene was found to include three exons which would encode a 421- (or 471-) amino acid (aa) polypeptide with a long hydrophobic stretch. The hh mRNA was about 2.3 kb long and expressed throughout development. The hh expression in an embryo occurred in stripes, while that in imaginal discs occurred in the posterior compartment. As a whole, the spatial expression pattern of hh mRNA was very similar to that of engrailed (en), a homeobox gene required for the formation of the anterior-posterior compartment boundary. Unlike en, no hh expression was observed in the central nervous system.
Asunto(s)
Comunicación Celular/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas de la Membrana/genética , Proteínas/genética , Animales , Secuencia de Bases , Northern Blotting , Comunicación Celular/fisiología , Clonación Molecular , ADN , Drosophila melanogaster/embriología , Proteínas Hedgehog , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Proteínas/fisiologíaRESUMEN
The fibroblast growth factor (FGF)/receptor system is thought to mediate various developmental events in vertebrates. We examined molecular structures and expression of DFR1 and DFR2, two Drosophila genes closely related to vertebrate FGF-receptor genes. DFR1 and DFR2 proteins contain two and five immunoglobulin-like domains, respectively, in the extracellular region, and a split tyrosine kinase domain in the intracellular region. In early embryos, DFR1 RNA expression, requiring both twist and snail proteins, is specific to mesodermal primordium and invaginated mesodermal cells. At later stages, putative muscle precursor cells and cells in the central nervous system (CNS) express DFR1. DFR2 expression occurs in endodermal precursor cells, CNS midline cells and certain ectodermal cells such as those of trachea and salivary duct. FGF-receptor homologues in Drosophila would thus appear essential for generation of mesodermal and endodermal layers, invaginations of various types of cells, and CNS formation.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Mesodermo/fisiología , Proteínas Tirosina Quinasas/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sistema Nervioso Central/embriología , Drosophila melanogaster/embriología , Endodermo/fisiología , Datos de Secuencia MolecularRESUMEN
BarH1 and BarH2 are two closely related homeo box genes that form a small complex at the Bar locus on the X chromosome of Drosophila. By immunostaining, we showed that BarH1 and BarH2 proteins are coexpressed in cells belonging to the central and peripheral nervous systems in embryos. In external sensory (es) organs, their expression was particularly apparent in thecogens (glial cells) and neurons at late development. Although deletion of BarH2 caused no appreciable morphological change in es organs, the simultaneous deletion of BarH1 and BarH2 led to a homeotic change in these organs with consequent conversion from campaniform-like sensilla to trichoid sensilla. In contrast, the overexpression of either BarH1 or BarH2 resulted in opposite morphological change. It would thus follow that BarH1 and BarH2 are a pair of redundant homeo box genes required for the subtype specification of es organs.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/embriología , Proteínas del Ojo/genética , Genes Homeobox/genética , Larva/genética , Células Fotorreceptoras/embriología , Factores de Transcripción , Animales , Drosophila melanogaster/genética , Proteínas de Homeodominio , Inmunohistoquímica , Larva/crecimiento & desarrollo , Familia de Multigenes/genética , Hibridación de Ácido NucleicoRESUMEN
In the Bar mutation of Drosophila, ommatidial differentiation is known to be suppressed in the anterior portion of the eye. Our structural analysis shows that the Bar region contains a pair of homeo box genes, BarH1 and BarH2. These genes encode polypeptides similar in size and sequence and share a common homeo domain that is identical in sequence except for putative trans-activator-binding sites. We also show, by mosaic analysis and immunostaining with anti-BarH1/BarH2 antibodies, that BarH1 and BarH2 are not only specifically coexpressed but also functionally required in R1/R6 prephotoreceptors and primary pigment cells in developing ommatidia. In R1/R6, the expression of BarH1 and BarH2 appears to be regulated by rough and glass gene products. BarH1 and BarH2 proteins are essential to normal lens formation, formation of three types of pigment cells, and elimination of excess cells from mature ommatidia. Taken together, our results suggest that Bar homeo domain proteins may play key roles in the fate-determination processes of pigment cells and cone cells.
Asunto(s)
Diferenciación Celular/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Proteínas del Ojo/genética , Genes Homeobox/genética , Pupa/genética , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/genética , Drosophila melanogaster/embriología , Regulación de la Expresión Génica/genética , Proteínas de Homeodominio , Inmunohistoquímica , Datos de Secuencia Molecular , Mosaicismo/genética , Células Fotorreceptoras/embriología , Pupa/crecimiento & desarrolloRESUMEN
The Bar mutation B of Drosophila melanogaster and optic morphology mutation Om(1D) of Drosophila ananassae result in suppression of ommatidium differentiation at the anterior portion of the eye. Examinations was made to determine the genes responsible for these mutations. Both loci were found to share in common a different type of homeobox gene, which we call "BarH1." Polyptides encoded by D. melanogaster and D. ananassae BarH1 genes consist of 543 and 604 amino acids, respectively, with homeodomains identical in sequence except for one amino acid substitution. A unique feature of these homeodomains is that the phenylalanine residue in helix 3, conserved in all metazoan homeodomains so far examined, is replaced by a tyrosine residue. By Northern blotting, considerably more BarH1 RNA was detected in the Bar mutant than in wild type. P element-mediated transformation showed Bar-like eye malformation to be induced by transient overexpression of the BarH1 gene in the late third-instar larvae. Somatic recombination analysis indicated normal gene functions of the Bar region, including the BarH1 gene, to be required for normal eye morphogenesis.
Asunto(s)
Drosophila melanogaster/genética , Drosophila/genética , Genes Homeobox , Mutación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Ojo/anatomía & histología , Ojo/crecimiento & desarrollo , Datos de Secuencia Molecular , Morfogénesis/genética , Hibridación de Ácido NucleicoRESUMEN
Following a corneal wound involving removal of the epithelium and basement membrane, the epithelium must migrate across bare stroma. To examine the effect of the removal of the basement membrane on epithelial migration and on protein and glycoprotein synthesis in both the epithelium and stroma, we performed superficial keratectomies on rabbits and allowed the corneas to heal in organ culture. We then analyzed the following parameters: (1) rate of epithelial wound closure; (2) proteins synthesized during epithelial wound closure in both the epithelium and stroma using SDS-PAGE; and (3) presence of fibronectin in the epithelium and stroma using immunodot blots and immunofluorescence. We found that: (1) a 7 mm keratectomy wound heals in 66 hr with a maximal rate of epithelial migration of 0.83 mm2/hr; (2) four proteins, 400+K, 220K, 70K, and 58K, are present in the epithelium migrating to close the wound that are not seen in the control epithelium; (3) a 220K band is seen in the wounded stroma but not in control stroma; and (4) fibronectin represents 2% of the total protein in the stroma 66 hr post-keratectomy but less than 0.02% in wounded epithelium, unwounded epithelium, and unwounded stroma.
Asunto(s)
Córnea/metabolismo , Proteínas del Ojo/biosíntesis , Fibronectinas/análisis , Cicatrización de Heridas , Animales , Membrana Basal/metabolismo , Córnea/análisis , Córnea/citología , Córnea/fisiopatología , Córnea/cirugía , Sustancia Propia/metabolismo , Células Epiteliales , Epitelio/análisis , Epitelio/metabolismo , Técnicas Inmunológicas , ConejosRESUMEN
When stratified corneal rat epithelium becomes migratory in response to a wound, an increase in binding by the lectins concanavalin A (Con A) and wheat germ agglutinin (WGA) is seen. These lectins bind the membranes of the cells of the leading edge of the migrating sheet more intensely than normal epithelium and epithelium behind the leading edge, suggesting a change in cell surface properties during migration. In the present study, analysis of cell surface proteins using lactoperoxidase-catalyzed iodination followed by SDS-PAGE indicates the appearance of a 70 K protein in epithelium migrating to cover a wound. Con A-affinity chromatography shows that two bands, 70 K and 155 K, increase 4.0- and 2.9-fold, respectively, in epithelium that is migrating. WGA-binding glycoproteins increased 1.61-fold following wounding with the major band present at 155 K in SDS-PAGE. The data suggest that these glycoproteins are responsible for the increase in Con A and WGA binding to cell membranes in migratory corneal epithelium.