RESUMEN
The Nodal and Hedgehog signaling pathways influence dorsoventral patterning at all axial levels of the CNS, but it remains largely unclear how these pathways interact to mediate patterning. Here we show that, in zebrafish, Nodal signaling is required for induction of the homeobox genes nk2.1a in the ventral diencephalon and nk2.1b in the ventral telencephalon. Hedgehog signaling is also required for telencephalic nk2.1b expression but may not be essential to establish diencephalic nk2.1a expression. Furthermore, Shh does not restore ventral diencephalic development in embryos lacking Nodal activity. In contrast, Shh does restore telencephalic nk2.1b expression in the absence of Nodal activity, suggesting that Hedgehog signaling acts downstream of Nodal activity to pattern the ventral telencephalon. Thus, the Nodal pathway regulates ventral forebrain patterning through both Hedgehog signaling-dependent and -independent mechanisms.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Hipotálamo/metabolismo , Proteínas/metabolismo , Transducción de Señal/fisiología , Telencéfalo/metabolismo , Transactivadores , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Pez Cebra , Animales , Diencéfalo/crecimiento & desarrollo , Diencéfalo/metabolismo , Proteínas Hedgehog , Proteína Homeobox Nkx-2.2 , Hipotálamo/crecimiento & desarrollo , Datos de Secuencia Molecular , Proteína Nodal , Telencéfalo/crecimiento & desarrollo , Pez CebraRESUMEN
Bone morphogenetic proteins (Bmps) are key regulators of dorsoventral (DV) patterning. Within the ectoderm, Bmp activity has been shown to inhibit neural development, promote epidermal differentiation and influence the specification of dorsal neurons and neural crest. In this study, we examine the patterning of neural tissue in mutant zebrafish embryos with compromised Bmp signalling activity. We find that although Bmp activity does not influence anteroposterior (AP) patterning, it does affect DV patterning at all AP levels of the neural plate. Thus, we show that Bmp activity is required for specification of cell fates around the margin of the entire neural plate, including forebrain regions that do not form neural crest. Surprisingly, we find that Bmp activity is also required for patterning neurons at all DV levels of the CNS. In swirl/bmp2b(-) (swr(-)) embryos, laterally positioned sensory neurons are absent whereas more medial interneuron populations are hugely expanded. However, in somitabun(-) (sbn(-)) embryos, which probably retain higher residual Bmp activity, it is the sensory neurons and not the interneurons that are expanded. Conversely, in severely Bmp depleted embryos, both interneurons and sensory neurons are absent and it is the most medial neurons that are expanded. These results are consistent with there being a gradient of Bmp-dependent positional information extending throughout the entire neural and non-neural ectoderm.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Cresta Neural/embriología , Neuronas/fisiología , Animales , Proteínas Morfogenéticas Óseas/fisiología , Diencéfalo/embriología , Diencéfalo/metabolismo , Ectodermo/fisiología , Cresta Neural/fisiología , Fenotipo , Prosencéfalo/embriología , Transducción de Señal , Telencéfalo/embriología , Telencéfalo/metabolismo , Pez Cebra/embriologíaRESUMEN
Five novel genes homologous to the homeobox-containing genes Xanf-1 and Xanf-2 of Xenopus and Hesx-1/Rpx of mouse have been identified as a result of a PCR survey of cDNA in sturgeon, zebrafish, newt, chicken and human. Comparative analysis of the homeodomain primary structure of these genes revealed that they belong to a novel class of homeobox genes, which we name Anf. All genes of this class investigated so far have similar patterns of expression during early embryogenesis, characterized by maximal transcript levels being present at the anterior extremity of the main embryonic body axis. The data obtained also suggest that, despite considerable high structural divergence between their homeodomains, all known Anf genes may be orthologues, and thus represent one of the most quickly evolving classes of vertebrate homeobox genes.
Asunto(s)
Genes Homeobox , Proteínas de Homeodominio/química , Vertebrados/genética , Proteínas de Xenopus , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Pollos , Embrión de Mamíferos/fisiología , Embrión no Mamífero/fisiología , Evolución Molecular , Peces , Proteínas de Homeodominio/biosíntesis , Humanos , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Salamandridae , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Vertebrados/embriología , Pez CebraRESUMEN
The epiphysial region of the dorsal diencephalon is the first site at which neurogenesis occurs in the roof of the zebrafish forebrain. We show that the homeobox containing gene floating head (flh) is required for neurogenesis to proceed in the epiphysis. In flh- embryos, the first few epiphysial neurons are generated, but beyond the 18 somite stage, neuronal production ceases. In contrast, in masterblind- (mbl-) embryos, epiphysial neurons are generated throughout the dorsal forebrain. We show that mbl is required to prevent the expression of flh in dorsal forebrain cells rostral to the epiphysis. Furthermore, epiphysial neurons are not ectopically induced in mbl-/flh- embryos, demonstrating that the epiphysial phenotype of mbl- embryos is mediated by ectopic Flh activity. We propose a role for Flh in linking the signaling pathways that regulate regional patterning to the signaling pathways that regulate neurogenesis.
Asunto(s)
Eliminación de Gen , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Prosencéfalo/embriología , Factores de Transcripción/genética , Proteínas de Pez Cebra , Animales , Trasplante de Tejido Encefálico , Embrión no Mamífero/fisiología , Inducción Embrionaria , Trasplante de Tejido Fetal , Proteínas de Homeodominio/biosíntesis , Hibridación in Situ , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Prosencéfalo/citología , Factores de Transcripción/biosíntesis , Pez CebraRESUMEN
Pax6 and Pax2 are members of the Pax family of transcription factors that are both expressed in the developing visual system of zebrafish embryos. Pax6 protein is present in all cells that form the neural retina and pigment epithelium, whereas Pax2 is located primarily in cells that will give rise to the optic stalk. In this study, we have addressed the role of midline signalling in the regulation of Pax2 and Pax6 distributions and in the subsequent morphogenesis of the eyes. Midline signalling is severely perturbed in cyclops mutant embryos resulting in an absence of ventral midline CNS tissue and fusion of the eyes. Mutant embryos ectopically express Pax6 in a bridge of tissue around the anterior pole of the neural keel in the position normally occupied by cells that form the optic stalks. In contrast, Pax2 protein is almost completely absent from this region in mutant embryos. Concommitant with the changes in Pax protein distribution, cells in the position of the optic stalks differentiate as retina. These results suggest that a signal emanating from the midline, which is absent in cyclops mutant embryos, may be required to promote Pax2 and inhibit Pax6 expression in cells destined to form the optic stalks. Sonic hedgehog (Shh also known as Vhh-1 and Hhg-1) is a midline signalling molecule that is absent from the neuroepithelium of cyclops mutant embryos at early developmental stages. To test the possibility that Shh might be able to regulate the spatial expression of Pax6 and Pax2 in the optic primordia, it was overexpressed in the developing CNS. The number of cells containing Pax2 was increased following shh overexpression and embryos developed hypertrophied optic stalk-like structures. Complimentary to the changes in Pax2 distribution, there were fewer Pax6-containing cells and pigment epithelium and neural retina were reduced. Our results suggest that Shh or a closely related signalling molecule emanating from midline tissue in the ventral forebrain either directly or indirectly induces the expression of Pax2 and inhibits the expression of Pax6 and thus may regulate the partitioning of the optic primordia into optic stalks and retinal tissue.
Asunto(s)
Proteínas de Unión al ADN/genética , Ojo/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Transducción de Señal , Transactivadores , Factores de Transcripción/genética , Pez Cebra/embriología , Animales , Proteínas del Ojo , Expresión Génica , Proteínas Hedgehog , Inmunohistoquímica , Hibridación in Situ , Morfogénesis/genética , Factor de Transcripción PAX2 , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas/genética , Proteínas Represoras , Pez Cebra/genética , Proteínas de Pez CebraRESUMEN
We have isolated zebrafish nk2.2, a member of the Nk-2 family of homeobox genes. nk2.2 is expressed in a continuous narrow band of cells along a boundary zone demarcating the location at which two of the earliest nuclei in the brain differentiate. This band of cells is located within a few cell diameters of cells expressing the signalling molecule sonic hedgehog/vertebrate hedgehog-1 (shh/vhh-1). Injection of shh/vhh-1 RNA results in ectopic expression of nk2.2 and concomitant abnormalities in the forebrain and eyes. Moreover, cyclops mutant embryos, which initially lack neurectodermal expression of shh/vhh-1, show a concomitant lack of nk2.2 expression. Together, these results suggest a requirement of shh/vhh-1 protein for the spatial regulation of nk2.2 expression.
Asunto(s)
Encéfalo/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Homeobox , Proteínas de Homeodominio/genética , Proteínas/genética , Transactivadores , Proteínas de Pez Cebra , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Diferenciación Celular , Ojo/embriología , Técnicas de Transferencia de Gen , Proteínas Hedgehog , Proteína Homeobox Nkx-2.2 , Hibridación in Situ , Datos de Secuencia Molecular , Neuronas/citología , Neuronas/fisiologíaRESUMEN
During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Neuronas/citología , Prosencéfalo/embriología , Proteínas de Pez Cebra , Animales , Diferenciación Celular , Proteínas de Unión al ADN/genética , Proteínas del Ojo , Factores de Transcripción Forkhead , Genes Reguladores , Hibridación in Situ , Morfogénesis , Vías Nerviosas/embriología , Factor de Transcripción PAX2 , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Represoras , Factores de Transcripción/genética , Proteínas Wnt , Pez CebraRESUMEN
We compared traditional complement fixation (CF) with a new passive agglutination method, the Seradyn Color Vue (SCV) test (Seradyn, Indianapolis, Ind.), for detection of Mycoplasma pneumoniae antibodies in 170 stored serum samples. The SCV test was 90% sensitive in identifying as positive 27 of 30 CF high-titer (> or = 1:64) serum samples and 100% specific in identifying as negative 134 of 134 CF low-titer (< or = 1:32) or negative (< 1:8) serum samples. The SCV test was technically undemanding, and it required no expensive equipment.
Asunto(s)
Pruebas de Aglutinación , Anticuerpos Antibacterianos/sangre , Pruebas de Fijación del Complemento , Mycoplasma pneumoniae/inmunología , Humanos , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
Using Immuno-Mycologics (IMMY; Norman, Okla.) histoplasmal yeast (HY) and mycelial (HM) antibody complement fixation test antigens, we retested 1,386 samples that were initially tested with Meridian Diagnostics, Inc. (Cincinnati, Ohio), antigens. Histoplasma antibody was identified (greater than or equal to 1:16) in 20% of HY and 5% of HM samples reported to have titers of less than 1:8 with Meridian reagents. IMMY titers were at least fourfold higher than Meridian titers in 39% of HY and 54% of HM samples that initially had titers of greater than or equal to 1:8 with Meridian antigens. Because 30 of 58 (52%) samples from confirmed cases of histoplasmosis yielded negative results with Meridian antigens and positive results upon retesting with IMMY antigens, we concluded that the Meridian antigens had less reactivity with human histoplasmal antibody.
Asunto(s)
Antígenos Fúngicos , Histoplasma/inmunología , Histoplasmosis/diagnóstico , Pruebas de Fijación del Complemento , Humanos , Sensibilidad y Especificidad , Serología/métodosRESUMEN
Four methods, latex agglutination, indirect fluorescent antibody, enzyme immunoassay, and complement fixation, were compared for cytomegalovirus antibody screening and for pre- and posttransplant determinations on bone marrow transplant recipients. Latex agglutination was most sensitive (98%) and specific (97%) for screening and pretransplant determinations and was quickest and easiest to perform. In posttransplant sera from allogeneic bone marrow transplant recipients, all methods except complement fixation detected cytomegalovirus antibody from therapeutically administered globulin preparations; this made it difficult to determine the significance of changes in cytomegalovirus antibody titer.
Asunto(s)
Anticuerpos Antivirales/análisis , Trasplante de Médula Ósea , Infecciones por Citomegalovirus/inmunología , Citomegalovirus/inmunología , Pruebas de Fijación del Complemento , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Pruebas de Fijación de Látex , Valor Predictivo de las PruebasRESUMEN
Phage T4 gene 49, encoding recombination endonuclease VII, specifies, by initiation from an AUG and an internal GUG codon, two in-frame overlapping peptides (of 18 and 12 kD). The gene is transcribed early and late, albeit from different promoters. The sequence predicts that in long early transcripts, initiated far upstream of the coding sequence, the Shine-Dalgarno sequence of the first ribosome binding site can be sequestered in a hairpin and/or cleaved. These processes might reduce initiation from the first AUG and facilitate initiation of the 12-kD peptide from the internal GUG. The potential of this hairpin to participate in Y structures or cruciforms suggests possible autoregulation. Shorter, more stable late transcripts initiated from a late promoter immediately upstream of the first ribosome binding site cannot form this hairpin. More efficient translation of the longer 18-kD gene 49 peptide from these late transcripts accounts for the strong dependence of endonuclease VII activity on late gene expression. An ORF downstream from gene 49 can be translated from a motA-dependent transcript that starts inside gene 49 as well as from the gene 49 transcripts. Its initiation codon overlaps the stop codon of gene 49, suggesting some coupling of translation. The deduced protein resembles, among others, the RexA protein of phage lambda. Possible implications for T4 recombination and for the interference of lambda lysogens with T4 gene 49 and rII mutants are discussed.