RESUMEN
The Gal4-UAS regulatory system of yeast is widely used to modulate gene expression in Drosophila; however, there are limitations to its usefulness in transgenic zebrafish, owing to progressive methylation and silencing of the CpG-rich multicopy upstream activation sequence. Although a modified, less repetitive UAS construct may overcome this problem, it is highly desirable to have additional transcriptional regulatory systems that can be applied independently or in combination with the Gal4/UAS system for intersectional gene expression. The Q transcriptional regulatory system of Neurospora crassa functions similarly to Gal4/UAS. QF is a transcriptional activator that binds to the QUAS upstream regulatory sequence to drive reporter gene expression. Unlike Gal4, the QF binding site does not contain essential CpG dinucleotide sequences that are subject to DNA methylation. The QS protein is a repressor of QF mediated transcriptional activation akin to Gal80. The functionality of the Q system has been demonstrated in Drosophila and Caenorhabditis elegans and we now report its successful application to a vertebrate model, the zebrafish, Danio rerio. Several tissue-specific promoters were used to drive QF expression in stable transgenic lines, as assessed by activation of a QUAS:GFP transgene. The QS repressor was found to dramatically reduce QF activity in injected zebrafish embryos; however, a similar repression has not yet been achieved in transgenic animals expressing QS under the control of ubiquitous promoters. A dual reporter construct containing both QUAS and UAS, each upstream of different fluorescent proteins was also generated and tested in transient assays, demonstrating that the two systems can work in parallel within the same cell. The adoption of the Q system should greatly increase the versatility and power of transgenic approaches for regulating gene expression in zebrafish.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Ingeniería Genética/métodos , Pez Cebra/genética , Animales , Animales Modificados Genéticamente/metabolismo , Regulación de la Expresión Génica/genética , Genes Fúngicos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Neurospora crassa/genética , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity, and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. Although many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions.
Asunto(s)
Habénula/embriología , Neurotransmisores/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Expresión Génica , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Habénula/metabolismo , Inmunohistoquímica , Neuronas/metabolismo , Neurotransmisores/genética , Especificidad de Órganos/genética , Fenotipo , Pez Cebra/genéticaRESUMEN
The nucleus incertus (NI), a conserved hindbrain structure implicated in the stress response, arousal, and memory, is a major site for production of the neuropeptide relaxin-3. On the basis of goosecoid homeobox 2 (gsc2) expression, we identified a neuronal cluster that lies adjacent to relaxin 3a (rln3a) neurons in the zebrafish analogue of the NI. To delineate the characteristics of the gsc2 and rln3a NI neurons, we used CRISPR/Cas9 targeted integration to drive gene expression specifically in each neuronal group, and found that they differ in their efferent and afferent connectivity, spontaneous activity, and functional properties. gsc2 and rln3a NI neurons have widely divergent projection patterns and innervate distinct subregions of the midbrain interpeduncular nucleus (IPN). Whereas gsc2 neurons are activated more robustly by electric shock, rln3a neurons exhibit spontaneous fluctuations in calcium signaling and regulate locomotor activity. Our findings define heterogeneous neurons in the NI and provide new tools to probe its diverse functions.
Asunto(s)
Neuronas , Pez Cebra , Animales , Neuronas/fisiología , Neuronas/metabolismo , Relaxina/metabolismo , Relaxina/genética , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Sistemas CRISPR-Cas , Rombencéfalo/fisiología , Rombencéfalo/metabolismoRESUMEN
Myeloid and plasmacytoid dendritic cells (DCs) are important mediators of both innate and adaptive immunity against pathogens such as HIV. During the course of HIV infection, blood DC numbers fall substantially. In the present study, we sought to determine how early in HIV infection the reduction occurs and whether the remaining DC subsets maintain functional capacity. We find that both myeloid DC and plasmacytoid DC levels decline very early during acute HIV infection. Despite the initial reduction in numbers, those DCs that remain in circulation retain their function and are able to stimulate allogeneic T-cell responses, and up-regulate maturation markers plus produce cytokines/chemokines in response to stimulation with TLR7/8 agonists. Notably, DCs from HIV-infected subjects produced significantly higher levels of cytokines/chemokines in response to stimulation with TLR7/8 agonists than DCs from uninfected controls. Further examination of gene expression profiles indicated in vivo activation, either directly or indirectly, of DCs during HIV infection. Taken together, our data demonstrate that despite the reduction in circulating DC numbers, those that remain in the blood display hyperfunctionality and implicates a possible role for DCs in promoting chronic immune activation.
Asunto(s)
Células Dendríticas/inmunología , Infecciones por VIH/inmunología , Recuento de Células Sanguíneas , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Estudios de Casos y Controles , Quimiocinas/biosíntesis , Quimiocinas/genética , Estudios Transversales , Citocinas/biosíntesis , Citocinas/genética , Células Dendríticas/clasificación , Expresión Génica , Infecciones por VIH/sangre , Infecciones por VIH/genética , Infecciones por VIH/virología , Humanos , Técnicas In Vitro , Estudios Longitudinales , Activación de Linfocitos , Transducción de Señal , Factores de Tiempo , Receptores Toll-Like/agonistas , Carga ViralRESUMEN
Decoding the functional connectivity of the nervous system is facilitated by transgenic methods that express a genetically encoded reporter or effector in specific neurons; however, most transgenic lines show broad spatiotemporal and cell-type expression. Increased specificity can be achieved using intersectional genetic methods which restrict reporter expression to cells that co-express multiple drivers, such as Gal4 and Cre. To facilitate intersectional targeting in zebrafish, we have generated more than 50 new Cre lines, and co-registered brain expression images with the Zebrafish Brain Browser, a cellular resolution atlas of 264 transgenic lines. Lines labeling neurons of interest can be identified using a web-browser to perform a 3D spatial search (zbbrowser.com). This resource facilitates the design of intersectional genetic experiments and will advance a wide range of precision circuit-mapping studies.
Asunto(s)
Mapeo Encefálico/métodos , Encéfalo/ultraestructura , Neuroimagen/métodos , Neuronas/ultraestructura , Animales , Animales Modificados Genéticamente/genética , Encéfalo/fisiología , Linaje de la Célula/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Integrasas/genética , Neuronas/fisiología , Factores de Transcripción/genética , Pez Cebra/genética , Pez Cebra/fisiologíaRESUMEN
The SaPIs are 15- to 17-kb mobile pathogenicity islands in staphylococci. They usually carry two or more superantigens and are responsible for most superantigen-related human diseases, especially staphylococcal toxic shock syndrome. SaPIs are extremely common in Staphylococcus aureus, with all but one of the sequenced genomes containing one or more. The SaPIs have a highly conserved overall genome organization, parallel to that of typical temperate phages. Each occupies a specific chromosomal site from which it is induced to excise and replicate by one or more specific staphylococcal phages. Following replication, the SaPI DNA is efficiently encapsidated into infectious small-headed phage-like particles, resulting in extremely high transfer frequencies.
Asunto(s)
Islas Genómicas , Staphylococcus/genética , Evolución Molecular , Genoma Bacteriano , Islas Genómicas/genética , Islas Genómicas/fisiología , Lisogenia , Recombinación Genética , Staphylococcus/patogenicidad , Fagos de Staphylococcus/genética , Superantígenos/genética , Terminología como AsuntoRESUMEN
SaPIs are a family of homologous phage-related pathogenicity islands in staphylococci that carry superantigen and other virulence genes, and are responsible for a wide variety of superantigen-related diseases. SaPIs are induced to excise and replicate by particular staphylococcal phages and are encapsidated in infectious, small-headed, phage-like particles, which are transmitted at very high frequency among staphylococcal strains and species. SaPI2 is a prototypical member of this family that was identified in a typical menstrual toxic shock syndrome (TSS) strain of Staphylococcus aureus, the so-called Harrisburg strain, and found to be mobilizable by typing phage 80. Most menstrual TSS strains belong to a highly uniform agr group III clone of electrophoretic type (ET) 41, and this study was undertaken to determine whether such strains typically carry SaPI2, and whether it has spread beyond the ET41 clone. We report here the complete sequence of SaPI2, describe its relation to other known SaPIs, and show that it, or a very similar element, is carried by most ET41 strains but that it has disseminated to other strains that have also been implicated in TSS. We show additionally, that SaPIs are widespread among the staphylococci and that most TSS strains carry two or more, including SaPI2.