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1.
Methods Cell Biol ; 133: 105-23, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27263410

RESUMEN

This chapter introduces the principles and advantages of selective plane illumination microscopy (SPIM) and compares it to commonly used epifluorescence or confocal setups. Due to the low phototoxicity, speed of imaging, high penetration depth, and spatiotemporal resolution, SPIM is predestined for in vivo imaging but can as well be used for in toto analysis of large fixed samples. Key points of light-sheet microscopy are highlighted and discussed priming the investigator to choose the best suitable system from the large collection of possible SPIM setups. Mounting of samples is shown and the demands for data acquisition, processing, handling, and visualization are discussed.


Asunto(s)
Microscopía Fluorescente/métodos , Oryzias/anatomía & histología , Retina/citología , Animales , Embrión no Mamífero/citología , Oryzias/crecimiento & desarrollo
2.
Oncogene ; 34(46): 5729-38, 2015 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-25728675

RESUMEN

P53 is an important tumor suppressor that, upon activation, induces growth arrest and cell death. Control of p53 is thus of prime importance for proliferating cells, but also for cancer therapy, where p53 activity contributes to the eradication of tumors. Mdm2 functionally inhibits p53 and targets the tumor suppressor protein for degradation. In a genetic screen, we identified TRIM25 as a novel regulator of p53 and Mdm2. TRIM25 increased p53 and Mdm2 abundance by inhibiting their ubiquitination and degradation in 26 S proteasomes. TRIM25 co-precipitated with p53 and Mdm2 and interfered with the association of p300 and Mdm2, a critical step for p53 polyubiquitination. Despite the increase in p53 levels, p53 activity was inhibited in the presence of TRIM25. Downregulation of TRIM25 resulted in an increased acetylation of p53 and p53-dependent cell death in HCT116 cells. Upon genotoxic insults, TRIM25 dampened the p53-dependent DNA damage response. The downregulation of TRIM25 furthermore resulted in massive apoptosis during early embryogenesis of medaka, which was rescued by the concomitant downregulation of p53, demonstrating the functional relevance of the regulation of p53 by TRIM25 in an organismal context.


Asunto(s)
Oryzias/embriología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Daño del ADN , Células HCT116 , Humanos , Células MCF-7 , Oryzias/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitinación
3.
Gene ; 322: 57-66, 2003 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-14644497

RESUMEN

We tested the Sleeping Beauty transposable element for its ability to efficiently insert transgenes into the genome of medaka (Oryzias latipes), an important model system for vertebrate development. We show that the SB transposon efficiently mediates integration of a reporter gene into the fish germ line. In pilot experiments, we established 174 transgenic lines with a transgenesis efficiency of 32%. Transgenes are stably transmitted to, and expressed in, subsequent generations. Interestingly, the transgenic lines show novel expression patterns with temporal and spatial specificity at a rate of 12% (21/174), likely due to both, enhancing and silencing position effects. Furthermore, promoter-dependent GFP expression in injected fish embryos is tightly correlated with germ line transmission, facilitating easy selection of founder fish. Thus, the SB transposon/transposase system provides a highly efficient tool for transgenesis in general and for the generation of novel reporter gene expression patterns in particular.


Asunto(s)
Elementos Transponibles de ADN/genética , Mutagénesis Insercional/métodos , Oryzias/genética , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Southern Blotting , ADN/química , ADN/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microinyecciones , Microscopía Fluorescente , Datos de Secuencia Molecular , Oryzias/embriología , Plásmidos/administración & dosificación , Plásmidos/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN
4.
Philos Trans R Soc Lond B Biol Sci ; 356(1414): 1545-63, 2001 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-11604122

RESUMEN

The shared roles of Pax6 and Six homologues in the eye development of various bilaterians suggest that Urbilateria, the common ancestors of all Bilateria, already possessed some simple form of eyes. Here, we re-address the homology of bilaterian cerebral eyes at the level of eye anatomy, of eye-constituting cell types and of phototransductory molecules. The most widespread eye type found in Bilateria are the larval pigment-cup eyes located to the left and right of the apical organ in primary, ciliary larvae of Protostomia and Deuterostomia. They can be as simple as comprising a single pigment cell and a single photoreceptor cell in inverse orientation. Another more elaborate type of cerebral pigment-cup eyes with an everse arrangement of photoreceptor cells is found in adult Protostomia. Both inverse larval and everse adult eyes employ rhabdomeric photoreceptor cells and thus differ from the chordate cerebral eyes with ciliary photoreceptors. This is highly significant because on the molecular level we find that for phototransduction rhabdomeric versus ciliary photoreceptor cells employ divergent rhodopsins and non-orthologous G-proteins, rhodopsin kinases and arrestins. Our comparison supports homology of cerebral eyes in Protostomia; it challenges, however, homology of chordate and non-chordate cerebral eyes that employ photoreceptor cells with non-orthologous phototransductory cascades.


Asunto(s)
Ojo/anatomía & histología , Fenómenos Fisiológicos Oculares , Células Fotorreceptoras de Invertebrados/fisiología , Filogenia , Animales , Axones , Evolución Biológica , Cordados no Vertebrados/fisiología , Larva , Células Fotorreceptoras de Vertebrados , Pigmentos Biológicos/fisiología , Transducción de Señal
5.
Development ; 128(20): 4035-44, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11641226

RESUMEN

The complete absence of eyes in the medaka fish mutation eyeless is the result of defective optic vesicle evagination. We show that the eyeless mutation is caused by an intronic insertion in the Rx3 homeobox gene resulting in a transcriptional repression of the locus that is rescued by injection of plasmid DNA containing the wild-type locus. Functional analysis reveals that Six3- and Pax6- dependent retina determination does not require Rx3. However, gain- and loss-of-function phenotypes show that Rx3 is indispensable to initiate optic vesicle evagination and to control vesicle proliferation, by that regulating organ size. Thus, Rx3 acts at a key position coupling the determination with subsequent morphogenesis and differentiation of the developing eye.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Ojo/crecimiento & desarrollo , Proteínas de Peces , Oryzias/crecimiento & desarrollo , Oryzias/genética , Retina/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Datos de Secuencia Molecular , Mutación , Proteínas del Tejido Nervioso/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas Represoras , Proteínas de Dominio T Box/genética , Temperatura , Proteína Homeobox SIX3
7.
Dev Genes Evol ; 211(7): 338-49, 2001 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11466530

RESUMEN

We report the identification and characterisation of five different Nkx5-related genes in medaka fish (Oryzias latipes). They constitute homologues of genes previously isolated in higher vertebrates, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3 and SOHo-1, and were named accordingly: OlNkx5--1.1, OlNkx5--2, OlNkx5--3 and OlSOHo. For the Nkx5--1 gene a new, second homologue, OlNkx5--1.2, was isolated. In medaka, Nkx5 and SOHo genes are differentially expressed in three developing sensory organs: eye, ear and lateral line and later in defined brain regions. Phylogenetic analyses of the entire Nkx5 family revealed that four paralogous Nkx5 groups, Nkx5--1, Nkx5--2, Hmx1/Nkx5--3/GH6 and SOHo, are present in vertebrates. Only some of the Nkx5 family members have been identified in singular vertebrate species so far. Here we present, for the first time, the isolation of representatives of each Nkx5 subgroup in one species, the medaka fish. Based on similarities in sequence and expression patterns, and genomic organisation we propose a model of the evolutionary history of the Nkx5 family. The model predicts that the four vertebrate Nkx5 genes arose by a tandem duplication, followed by chromosomal duplication. The two Nkx5--1 genes identified so far exclusively in medaka most probably result from an additional genome duplication in the fish lineage.


Asunto(s)
Oído/embriología , Evolución Molecular , Ojo/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Familia de Multigenes , Proteínas del Tejido Nervioso/genética , Oryzias/genética , Secuencia de Aminoácidos , Animales , Ojo/embriología , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
8.
Nature ; 409(6816): 81-5, 2001 Jan 04.
Artículo en Inglés | MEDLINE | ID: mdl-11343117

RESUMEN

Bilateria are subdivided into Protostomia and Deuterostomia. Indirect development through primary, ciliary larvae occurs in both of these branches; however, the closing blastopore develops into mouth and anus in Protostomia and into anus only in Deuterostomia. Because of this important difference in larval gut ontogeny, the tube-shaped guts in protostome and deuterostome primary larvae are thought to have evolved independently. To test this hypothesis, we have analysed the expression of brachyury, otx and goosecoid homologues in the polychaete Platynereis dumerilii, which develops by means of a trochophora larva-the primary, ciliary larva prototypic for Protostomia. Here we show that brachyury expression in the ventral portion of the developing foregut in Platynereis and also otx expression along ciliated bands in the mouth region of the trochophora larva parallels expression in primary larvae in Deuterostomia. In addition, goosecoid expression in the foregut of Platynereis mirrors the function in higher Deuterostomia. We present molecular evidence for the evolutionary conservation of larval foreguts and mouth regions of Protostomia and Deuterostomia. Our data indicate that Urbilateria, the common bilaterian ancestors, developed through a primary, ciliary larva that already possessed a tripartite tube-shaped gut.


Asunto(s)
Evolución Biológica , Tipificación del Cuerpo , Proteínas Fetales , Poliquetos/clasificación , Proteínas Represoras , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Clonación Molecular , Evolución Molecular , Expresión Génica , Proteína Goosecoide , Proteínas de Homeodominio/genética , Intestinos/anatomía & histología , Larva/anatomía & histología , Datos de Secuencia Molecular , Boca/anatomía & histología , Factores de Transcripción Otx , Filogenia , Poliquetos/anatomía & histología , Poliquetos/genética , Homología de Secuencia de Aminoácido , Proteínas de Dominio T Box/genética
9.
Mech Dev ; 97(1-2): 133-9, 2000 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11025214

RESUMEN

In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.


Asunto(s)
Oryzias/embriología , Oryzias/genética , Animales , Ojo/crecimiento & desarrollo , Femenino , Técnicas Genéticas , Masculino , Mutagénesis
10.
Mech Dev ; 95(1-2): 175-87, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10906460

RESUMEN

The induction of sensory organ placodes, in particular the lens placode, represents the paradigm for induction. We show that medaka Sox3 is expressed in the neuroectoderm and in the placodes of all sensory organs prior to placode formation and subsequently in placode-derived tissues. Ectopic Sox3 expression leads to ectopic expression of Pax6 and Eya1 in embryonic ectoderm and causes ectopic lens and otic vesicle formation. The descendants of cells ectopically expressing Sox3-mRNA contribute to ectopic lens tissue. This suggests a permissive role for Sox3 in establishing a placodal competence. In addition, ectopic Sox3 expression leads to the dysgenesis of the endogenous sensory organs. Both effects of ectopic Sox3 expression can be separated by ectopic expression of a truncated Sox3 variant depending on its expression level. Our data suggests that Sox3 is a permissive factor for sensory placode formation and plays an important role in sensory organ development.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Cristalino/embriología , Oryzias/embriología , Secuencia de Aminoácidos , Animales , Regulación del Desarrollo de la Expresión Génica , Cristalino/fisiología , Datos de Secuencia Molecular , Morfogénesis , Oryzias/fisiología , Factores de Transcripción SOXB1 , Alineación de Secuencia , Factores de Transcripción/fisiología
11.
Development ; 127(9): 1911-9, 2000 May.
Artículo en Inglés | MEDLINE | ID: mdl-10751179

RESUMEN

In early vertebrate eye development, the retinal anlage is specified in the anterior neuroectoderm. During neurulation, the optic vesicles evaginate from the lateral wall of the prosencephalon. Here we describe the temperature-sensitive mutation eyeless in the Japanese medakafish. Marker gene analysis indicates that, whereas, specification of two retinal primordia and proximodistal patterning takes place in the mutant embryo, optic vesicle evagination does not occur and subsequent differentiation of the retinal primordia is not observed. The mutation eyeless thus uncouples patterning and morphogenesis at early steps of retinal development. Temperature-shift experiments indicate a requirement for eyeless activity prior to optic vesicle evagination. Cell transplantation shows that eyeless acts cell autonomously.


Asunto(s)
Tipificación del Cuerpo/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Ojo/embriología , Proteínas de Homeodominio , Oryzias/embriología , Animales , Apoptosis/genética , Encéfalo/embriología , División Celular , Proteínas del Ojo , Regulación del Desarrollo de la Expresión Génica , Genes Recesivos , Hibridación in Situ , Morfogénesis , Mutación , Oryzias/genética , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas de Plantas/genética , Proteínas Represoras , Retina/embriología , Temperatura
12.
Bioorg Med Chem ; 8(2): 405-12, 2000 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-10722163

RESUMEN

N-Nitroso-N-oxybenzenamine ammonium salts with -OMe, -Me, -H, -F, -Cl, -CF3, and -SO2Me substituents at the para position of the phenyl ring constitute a new class of-redox sensitive nitric oxide (NO) releasing compounds. These compounds yield nitric oxide and the corresponding nitrosobenzene derivatives by a spontaneous dissociation mechanism after undergoing a one electron oxidation. Oxidation of these compounds can be achieved through chemical, electrochemical and enzymatic methods. It was observed electrochemically that the amount of NO generated was dependent on the substituent effect and the applied oxidation potential. Electron-withdrawing substituents increase the oxidation potential of the compound. A linear correlation was observed when the peak potentials for the oxidation were graphed versus the Hammett substituent constant. Density functional theory calculations were also performed on this series of compounds. The theoretical oxidation energies of the corresponding anions show a strong linear correlation with the experimental potentials. Furthermore, enzymatic oxidation using horseradish peroxidase showed a similar substituent effect. These results indicate that substitution at the para position of the phenyl ring has a profound effect on the stability, oxidation potential and enzymatic kinetic properties of the compounds. Thus para-substituted N-nitroso-N-oxybenzenamine salts comprise a new class of redox-sensitive nitric oxide releasing agents.


Asunto(s)
Donantes de Óxido Nítrico/farmacología , Compuestos de Amonio Cuaternario/farmacología , Electroquímica , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Donantes de Óxido Nítrico/química , Oxidación-Reducción , Compuestos de Amonio Cuaternario/química
13.
Dev Genes Evol ; 210(1): 28-33, 2000 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-10603084

RESUMEN

To rapidly isolate genes specifically expressed during medaka development we generated a cDNA library enriched for genes expressed in the head region of the developing embryo. Clones were spotted on filters automatically and preselected for abundantly expressed genes by hybridizing them with a probe derived from RNA of undifferentiated totipotent cells. Of the nonhybridizing clones 153 were chosen randomly and further analyzed by whole-mount in situ hybridization. There were 67 selected clones differentially expressed in the developing embryos, and 48 of these were expressed in the developing head. Differentially expressed genes were either of novel type or showed homology to known genes containing DNA binding motifs or to putative housekeeping genes.


Asunto(s)
Antígenos de Diferenciación , Expresión Génica , Hibridación in Situ/métodos , Animales , ADN Complementario/análisis , ADN Complementario/genética , Biblioteca de Genes , Oryzias/genética
14.
Genetics ; 153(3): 1385-94, 1999 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-10545466

RESUMEN

The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus.


Asunto(s)
Ciprinodontiformes/genética , Elementos Transponibles de ADN , Enfermedades de los Peces/genética , Proteínas de Peces , Eliminación de Gen , Melanoma/veterinaria , Mutación , Oncogenes , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cruzamientos Genéticos , Melanoma/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Transcripción Genética
15.
Development ; 126(24): 5659-67, 1999 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-10572042

RESUMEN

Signalling by fibroblast growth factors (FGFs) at the mid-hindbrain boundary (MHB) is of central importance for anteroposterior neural patterning from the isthmic organiser. Graded suppression of FGF signalling by increasing amounts of a dominant negative FGF receptor provides evidence that in addition to anteroposterior patterning, FGF signalling is also involved in patterning along the dorsoventral axis at the MHB. FGF signalling at the MHB is required for the activation of the HH target gene spalt at the MHB. Our results indicate that FGF signalling mediates the competence of the MHB to activate spalt in response to SHH. This interdependence of the two signalling pathways is also found in the outbudding optic vesicle where HH requires functional FGF signalling to activate spalt in the proximal eye region.


Asunto(s)
Tipificación del Cuerpo , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Tirosina Quinasas , Rombencéfalo/embriología , Transducción de Señal , Transactivadores , Proteínas de Pez Cebra , Animales , Secuencia de Bases , Proteínas de Drosophila , Ojo/embriología , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Proteínas de Homeodominio/genética , Mesencéfalo/embriología , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Oryzias/embriología , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Receptores de Factores de Crecimiento de Fibroblastos/genética , Factores de Transcripción/genética , Proteínas Wnt
16.
J Comp Neurol ; 413(3): 385-404, 1999 Oct 25.
Artículo en Inglés | MEDLINE | ID: mdl-10502247

RESUMEN

We analyzed the medaka optic tectum (OT) morphogenesis by using 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry (with a new method we developed for pulse-labeling embryos) and in situ hybridization with three probes, two for recently cloned homeobox genes (Ol-Prx3 [Paired-Related-Homeobox3] and Ol-Gsh1 [Genetic-Screen-Homeobox1]) and one for Ol-tailless. The tectal anlage first appears as a sheet of proliferating cells expressing Ol-Gsh1 and Ol-tailless but not Ol-Prx3. Cells subsequently cease to proliferate in a superficial and rostral zone and begin to express Ol-Prx3. When tectal lamination begins, the proliferative zone (mpz) becomes restricted to a crescent at the OT medial, caudal, and lateral margin. This mpz functions throughout the fish's entire life. It produces cells that are added at the OT's edge as radial rows, spanning every layer of the OT. The cells of the mpz continue to express Ol-tailless in the adult, whereas Ol-Gsh1 expression is turned off. When superficial layers form, Ol-Prx3 expression becomes restricted to the underlying deep layer, where it persists in the adult. Ol-Prx3 seems to be a marker for the differentiation of a subset of deep cells and allows analysis of tectal lamination, whereas Ol-tailless and Ol-Gsh1 could be involved in the control of tectal cell proliferation. This study constitutes a first step toward molecular approach to OT development in anamniotes. We compare and discuss the expression patterns of the homologs of the genes studied, and more generally the morphogenetic patterns of the medaka tectum, with those encountered in other cortical structures and in other vertebrate groups.


Asunto(s)
Neuronas/citología , Oryzias/embriología , Colículos Superiores/embriología , Animales , Evolución Biológica , División Celular , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/análisis , Proteínas de Homeodominio/genética , Morfogénesis , Oryzias/fisiología , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/genética , Colículos Superiores/citología
17.
Development ; 126(17): 3769-79, 1999 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-10433907

RESUMEN

In vertebrates, the engrailed genes are expressed at early neurula stage in a narrow stripe encompassing the midbrain-hindbrain boundary (MHB), a region from which a peculiar structure, the isthmus, is formed. Knock-out experiments in mice demonstrated that these genes are essential for the development of this structure and of its derivatives. In contrast, little is known about the effect of an overexpression of engrailed genes in vertebrate development. Here we report the isolation of Ol-eng2, a medaka fish (Oryzias latipes) engrailed gene. We have monitored the effects of its widespread expression following mRNA injections in 1- and 2-cell medaka and Xenopus embryos. We found that the ectopic expression of Ol-eng2 predominantly results in an altered development of the anterior brain, including an inhibition of optic vesicle formation. No change in the patterns of mesencephalic and telencephalic markers were observed. In contrast, expressions of markers of the diencephalon were strongly repressed in injected embryos. Furthermore, the endogenous Ol-eng2, Pax2, Wnt1 and Fgf8, which are essential components of the MHB genetic cascade, were ectopically expressed in this region. Therefore, we propose that Ol-eng2 induces de novo formation of an isthmus-like structure, which correlates with the development of ectopic midbrain structures, including optic tectum. A competence of the diencephalon to change to a midbrain fate has been demonstrated in isthmic graft experiments. Our data demonstrate that this change can be mimicked by ectopic engrailed expression alone.


Asunto(s)
Diencéfalo/embriología , Proteínas de Homeodominio/genética , Mesencéfalo/embriología , Proteínas del Tejido Nervioso/genética , Oryzias/embriología , Oryzias/genética , Rombencéfalo/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Tipificación del Cuerpo/genética , Cartilla de ADN/genética , ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Hibridación in Situ , Ratones , Microinyecciones , Datos de Secuencia Molecular , ARN Mensajero/administración & dosificación , ARN Mensajero/genética , Homología de Secuencia de Aminoácido
18.
Mech Dev ; 85(1-2): 15-25, 1999 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10415343

RESUMEN

We have previously cloned several members of the TGF-beta superfamily of growth factors in zebrafish, one of which, Radar, belongs to the Dpp-Vg1-related (DVR) subgroup, with highest homology to GDF6. The pattern of expression of Radar suggested a possible involvement in several induction steps during embryogenesis including in the dorsal neural tube, red blood cells, the dorsal fin and the retina. We have analyzed the pattern of expression of Radar in comparison with that of a marker of dorsal neural tube structures, msxC and show that Radar and msxC are expressed in similar and/or adjacent tissues throughout embryogenesis. In order to demonstrate a functional relationship between these two proteins, we have generated a full-length cDNA for Radar and shown that Radar overexpression by DNA injection maintains expression of msxC in tissues where it is normally expressed then turned off, in particular in the dorsal neurectoderm. Study of the phenotype of a mutant carrying a deletion of Radar shows a loss of identity and death of the cells of the dorsal neural tube. Taken together these results suggest that Radar could be involved in maintaining the identity of cells of the dorsal-most neural tube and of at least a subset of neural crest cells.


Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Ectodermo/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/embriología , Factor de Crecimiento Transformador beta/fisiología , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factor 6 de Diferenciación de Crecimiento , Sustancias de Crecimiento/genética , Datos de Secuencia Molecular , Pez Cebra/fisiología
19.
Genes Dev ; 13(6): 649-54, 1999 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-10090721

RESUMEN

The homeobox gene sine oculis (so) is essential for visual system formation in Drosophila. A vertebrate member of the so/Six gene family, Six3, is expressed in the developing eye and forebrain. Injection of Six3 RNA into medaka fish embryos causes ectopic Pax6 and Rx2 expression in midbrain and cerebellum, resulting in the formation of ectopic retinal primordia. Injected mouse Six3 RNA initiates ectopic expression of endogenous medaka Six3, uncovering a feedback control of Six3 expression. Initiation of ectopic retina formation reveals a pivotal role for Six3 in vertebrate retina development and hints at a conserved regulatory network underlying vertebrate and invertebrate eye development.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Retina/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Embrión no Mamífero/metabolismo , Proteínas del Ojo , Ratones , Oryzias/embriología , ARN/administración & dosificación , Retina/embriología , Transcripción Genética , Proteína Homeobox SIX3
20.
DNA Cell Biol ; 17(10): 835-47, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9809745

RESUMEN

Annexins are Ca2+-regulated membrane binding proteins implicated in a wide range of membrane-related and signal transduction events, including the endocytosis of membrane receptors and Ca2+-regulated as well as constitutive secretion. To date, 10 unique members of this multigene family have been identified in a variety of cell types and tissues of higher vertebrates, with different members showing distinct tissue distributions in the adult organisms. To establish whether annexins also function in embryonic development, we analyzed the expression pattern during vertebrate morphogenesis using the medaka fish Oryzias latipes as a model system. From a larval medaka cDNA library, we isolated four types of clones, which were shown by sequence analysis to encode four different annexins (herein referred to as max 1-4). A comparison with known annexin sequences in the databases revealed that two medaka annexins (max 1 and 2) are highly similar in sequence to mammalian annexins V and IV, respectively, whereas the other two medaka annexins (max 3 and 4) are probably novel members of the family most closely related to mammalian annexins I and XI. Using whole-mount RNA in situ hybridization, we showed that the expression of the different medaka annexins during embryogenesis was strictly regulated at both the spatial and the temporal level. High levels of max 1, 2, and 3 transcripts were present in the developing stomach, gut, liver, air-bladder, and rectum during somitogenesis, thus identifying the digestive tract as the prime region of annexin expression. Interestingly, two structures playing crucial roles in neuronal patterning showed a distinct expression of annexins. The mesendoderm of the anterior prechordal plate of neurula-stage embryos was a site of max 4 transcription, and the floor plate of somitogenesis-stage embryos showed expression of max 2 and 3 to differing rostrocaudal extends along the brain and spinal cord. These results suggest specific functions of different annexins during vertebrate morphogenesis.


Asunto(s)
Anexinas/genética , Regulación del Desarrollo de la Expresión Génica , Familia de Multigenes , Oryzias/genética , Envejecimiento , Secuencia de Aminoácidos , Animales , Anexinas/biosíntesis , Anexinas/química , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Embrión no Mamífero/fisiología , Biblioteca de Genes , Humanos , Larva , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Oryzias/embriología , Oryzias/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Sondas ARN , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Especificidad de la Especie , Transcripción Genética
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