RESUMEN
Segregation of the future germ line defines a crucial cell fate decision during animal development. In Xenopus, germ cells are specified by inheritance of vegetally localized maternal determinants, including a group of specific mRNAs. Here, we show that the vegetal localization elements (LE) of Xenopus Dead end (XDE) and of several other germ-line-specific, vegetally localized transcripts mediate germ cell-specific stabilization and somatic clearance of microinjected reporter mRNA in Xenopus embryos. The part of XDE-LE critical for somatic RNA clearance exhibits homology to zebrafish nanos1 and appears to be targeted by Xenopus miR-18 for somatic mRNA clearance. Xenopus Elr-type proteins of the vegetal localization complex can alleviate somatic RNA clearance of microinjected XDE-LE and endogenous XDE mRNA. ElrB1 synergizes with Xenopus Dead end protein in the stabilization of XDE-LE mRNA. Taken together, our findings unveil a functional link of vegetal mRNA localization and the protection of germ-line mRNAs from somatic clearance.
Asunto(s)
MicroARNs/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Animales , Secuencia de Bases , Proteína 2 Similar a ELAV , Embrión no Mamífero/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Células Germinativas/metabolismo , MicroARNs/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transcripción Genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genéticaRESUMEN
Successful establishment and persistence of adenovirus (Ad) infections are facilitated by immunosubversive functions encoded in the early transcription unit 3 (E3). The E3/19K protein has a dual role, preventing cell surface transport of MHC class I/HLA class I (MHC-I/HLA-I) Ags and the MHC-I-like molecules (MHC-I chain-related chain A and B [MICA/B]), thereby inhibiting both recognition by CD8 T cells and NK cells. Although some crucial functional elements in E3/19K have been identified, a systematic analysis of the functional importance of individual amino acids is missing. We now have substituted alanine for each of 21 aas in the luminal domain of Ad2 E3/19K conserved among Ads and investigated the effects on HLA-I downregulation by coimmunoprecipitation, pulse-chase analysis, and/or flow cytometry. Potential structural alterations were monitored using conformation-dependent E3/19K-specific mAbs. The results revealed that only a small number of mutations abrogated HLA-I complex formation (e.g., substitutions W52, M87, and W96). Mutants M87 and W96 were particularly interesting as they exhibited only minimal structural changes suggesting that these amino acids make direct contacts with HLA-I. The considerable number of substitutions with little functional defects implied that E3/19K may have additional cellular target molecules. Indeed, when assessing MICA/B cell-surface expression we found that mutation of T14 and M82 selectively compromised MICA/B downregulation with essentially no effect on HLA-I modulation. In general, downregulation of HLA-I was more severely affected than that of MICA/B; for example, substitutions W52, M87, and W96 essentially abrogated HLA-I modulation while largely retaining the ability to sequester MICA/B. Thus, distinct conserved amino acids seem preferentially important for a particular functional activity of E3/19K.
Asunto(s)
Infecciones por Adenoviridae/metabolismo , Proteínas E3 de Adenovirus/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Infecciones por Adenoviridae/genética , Proteínas E3 de Adenovirus/genética , Secuencia de Aminoácidos , Secuencia Conservada , Regulación hacia Abajo , Citometría de Flujo , Humanos , Inmunoprecipitación , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , TransfecciónRESUMEN
The adenovirus (Ad) early transcription unit 3 (E3) encodes multiple immunosubversive functions that are presumed to facilitate the establishment and persistence of infection. Indeed, the capacity of E3/19K to inhibit transport of HLA class I (HLA-I) to the cell surface, thereby preventing peptide presentation to CD8(+) T cells, has long been recognized as a paradigm for viral immune evasion. However, HLA-I downregulation has the potential to render Ad-infected cells vulnerable to natural killer (NK) cell recognition. Furthermore, expression of the immediate-early Ad gene E1A is associated with efficient induction of ligands for the key NK cell-activating receptor NKG2D. Here we show that while infection with wild-type Ad enhances synthesis of the NKG2D ligands, major histocompatibility complex class I chain-related proteins A and B (MICA and MICB), their expression on the cell surface is actively suppressed. Both MICA and MICB are retained within the endoplasmic reticulum as immature endoglycosidase H-sensitive forms. By analyzing a range of cell lines and viruses carrying mutated versions of the E3 gene region, E3/19K was identified as the gene responsible for this activity. The structural requirements within E3/19K necessary to sequester MICA/B and HLA-I are similar. In functional assays, deletion of E3/19K rendered Ad-infected cells more sensitive to NK cell recognition. We report the first NK evasion function in the Adenoviridae and describe a novel function for E3/19K. Thus, E3/19K has a dual function: inhibition of T-cell recognition and NK cell activation.
Asunto(s)
Proteínas E3 de Adenovirus/inmunología , Adenovirus Humanos/inmunología , Compartimento Celular , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/inmunología , Adenovirus Humanos/química , Expresión Génica , Inmunidad , Células Asesinas Naturales/virología , Ligandos , Receptores de Células Asesinas Naturales , Linfocitos T/inmunologíaRESUMEN
The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25 kDa transmembrane glycoprotein binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide-MHC complexes to the cell surface and consequently T cell recognition. Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress natural killer (NK) cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3-13, 15-21 and 41-45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed. Secondary structure prediction suggests that the lumenal domain contains six beta-strands and an alpha-helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition.
Asunto(s)
Proteínas E3 de Adenovirus/química , Proteínas E3 de Adenovirus/inmunología , Anticuerpos Monoclonales/inmunología , Mutagénesis , Alanina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Especificidad de Anticuerpos/inmunología , Unión Competitiva , Carbohidratos/química , Línea Celular , Secuencia Conservada , Cisteína/genética , Epítopos/química , Epítopos/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/inmunología , Mutación/genética , Mapeo Peptídico , Conformación Proteica , Proteínas Recombinantes/inmunologíaRESUMEN
The Xenopus germ line is derived from a specialized region in the vegetal hemisphere of the oocyte, the germ plasm. Several maternal transcripts harboured in this region have been connected to the process of germ cell specification. We identified and functionally characterized a novel vegetally localizing mRNA encoding a glutamate receptor interacting protein (GRIP) family member in Xenopus, termed XGRIP2.1. XGRIP2.1 is specifically associated with the germ plasm and PGCs throughout Xenopus embryogenesis. Morpholino-mediated knockdown and overexpression of a putative dominant negative XGRIP2.1 protein fragment reduced average PGC numbers and interfered with the proper anteroposterior positioning of PGCs at tailbud stages. Thus, our results suggest that XGRIP2.1 is required for normal PGC development and migration in Xenopus.
Asunto(s)
Tipificación del Cuerpo , Proteínas Portadoras , Células Germinativas/fisiología , ARN Mensajero Almacenado/metabolismo , Proteínas de Xenopus , Xenopus laevis/anatomía & histología , Xenopus laevis/embriología , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Movimiento Celular , Supervivencia Celular , Marcación de Gen , Células Germinativas/citología , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero Almacenado/genética , Ratas , Alineación de Secuencia , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/crecimiento & desarrolloRESUMEN
Xenopus embryos provide a powerful model system to investigate the complex molecular mechanisms, which are controlled by or control the activity of the Hedgehog (Hh) signaling pathway. The use of synthetic mRNA or antisense oligonucleotide (morpholino) microinjection into blastomeres of early embryos or by simply treating the embryos with small organic inhibitors, has already led to an idea of the network in which the Hh pathway is embedded. More needs to be done in order to achieve a detailed understanding of how the different players of the Hh signaling pathway are integrated to control different genetic programs, such as axis formation in early embryos or cell differentiation during retinogenesis.
Asunto(s)
Embrión no Mamífero/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Microinyecciones , Biología Molecular/métodos , Transducción de Señal , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Animales , Bioensayo , Embrión no Mamífero/efectos de los fármacos , Lovastatina/farmacología , Fenotipo , ARN Mensajero/administración & dosificación , Transducción de Señal/efectos de los fármacos , Alcaloides de Veratrum/farmacologíaRESUMEN
Analysis of spatiotemporal patterns of gene expression is an important prerequisite for understanding the molecular basis of embryogenesis. Tissue-specific resolution is desirable, but often not achieved owing to methodical limitations. We used a common model system for embryonic development--the South African clawed frog Xenopus laevis--to demonstrate that laser microdissection and laser-mediated catapulting of tissue samples from histologic sections are feasible even for yolk-rich, fragile embryonic tissue. A combination with RT-PCR provides the possibility of detecting tissue-specific gene expression with high resolution and fidelity. We show that specimens of various sizes and shapes can easily be procured by laser microdissection and pressure catapulting (LMPC). Subsequent RNA-isolation and nested RT-PCR for marker genes revealed that the combination of these methods allows for analysis of specific gene expression in micro-areas. We report on the efficiency and reliability of detection of marker genes in dissected tissue. We further discuss the question of whether such a combination can be applied to certain issues raised in developmental biology with regard to other techniques.
Asunto(s)
Disección/métodos , Expresión Génica , Rayos Láser , Xenopus laevis/embriología , Animales , Cartilla de ADN , Embrión no Mamífero , ARN/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We have isolated two related Xenopus homologues of the homeotic zinc finger protein Teashirt1 (Tsh1), XTsh1a and XTsh1b. While Drosophila teashirt specifies trunk identity in the fly, the developmental relevance of vertebrate Tsh homologues is unknown. XTsh1a/b are expressed in prospective trunk CNS throughout early neurula stages and later in the migrating cranial neural crest (CNC) of the third arch. In postmigratory CNC, XTsh1a/b is uniformly activated in the posterior arches. Gain- and loss-of-function experiments reveal that reduction or increase of XTsh1 levels selectively inhibits specification of the hindbrain and mid/hindbrain boundary in Xenopus embryos. In addition, both overexpression and depletion of XTsh1 interfere with the determination of CNC segment identity. In transplantation assays, ectopic XTsh1a inhibits the routing of posterior, but not of mandibular CNC streams. The loss of function phenotype could be rescued with low amounts either of XTsh1a or murine Tsh3. Our results demonstrate that proper expression of XTsh1 is essential for segmentally restricted gene expression in the posterior brain and CNC and suggest for the first time that teashirt genes act as positional factors also in vertebrate development.
Asunto(s)
Encéfalo/embriología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Cresta Neural/embriología , Cráneo/embriología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Animales , Tipificación del Cuerpo , Encéfalo/metabolismo , Movimiento Celular , Embrión no Mamífero , Proteínas de Homeodominio/fisiología , Modelos Biológicos , Cresta Neural/metabolismo , Cresta Neural/fisiología , Cresta Neural/trasplante , Isoformas de Proteínas , Cráneo/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMEN
Secreted proteins of the Hedgehog (Hh) family direct the development of diverse organs and tissues of vertebrates and invertebrates. Gli-type zinc finger proteins function as transcriptional mediators of the Hh signaling cascade and were implicated both in the activation and repression of Hh target genes. The differential activity of Gli-type zinc finger proteins is regulated on the level of proteolytic processing and subcellular localization as a complex concert of Hh-responsive, intracellular determinants. Here, we provide a survey of recent studies on the characterization of molecular mechanisms involved in the interpretation of Hh signals by Gli-type zinc finger proteins.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas Oncogénicas/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Hedgehog , Humanos , Proteínas Oncogénicas/genética , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1 , Dedos de ZincRESUMEN
Vertebrate inner ear development is initiated by the specification of the otic placode, an ectodermal structure induced by signals from neighboring tissue. Although several signaling molecules have been identified as candidate otic inducers, many details of the process of inner ear induction remain elusive. Here, we report that otic induction is responsive to the level of Hedgehog (Hh) signaling activity in Xenopus, making use of both gain- and loss-of-function approaches. Ectopic activation of Hedgehog signaling resulted in the development of ectopic vesicular structures expressing the otic marker genes XPax-2, Xdll-3, and Xwnt-3A, thus revealing otic identity. Induction of ectopic otic vesicles was also achieved by misexpression of two different inhibitors of Hh signaling: the putative Hh antagonist mHIP and XPtc1deltaLoop2, a dominant-negative form of the Hh receptor Patched. In addition, misexpression of XPtc1deltaLoop2 as well as treatment of Xenopus embryos with the specific Hh signaling antagonist cyclopamine resulted in the formation of enlarged otic vesicles. In summary, our observations suggest that a defined level of Hh signaling provides a restrictive environment for otic fate in Xenopus embryos.
Asunto(s)
Oído Interno/embriología , Ectodermo/metabolismo , Receptores Acoplados a Proteínas G , Transducción de Señal , Transactivadores/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriología , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Oído Interno/metabolismo , Embrión no Mamífero , Inducción Embrionaria , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Factor de Transcripción PAX2 , Proteínas/genética , Proteínas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , Proteínas de Xenopus/genéticaRESUMEN
Sonic hedgehog is involved in eye field separation along the proximodistal axis. We show that Hh signalling continues to be important in defining aspects of the proximodistal axis as the optic vesicle and optic cup mature. We show that two other Hedgehog proteins, Banded hedgehog and Cephalic hedgehog, related to the mouse Indian hedgehog and Desert hedgehog, respectively, are strongly expressed in the central retinal pigment epithelium but excluded from the peripheral pigment epithelium surrounding the ciliary marginal zone. By contrast, downstream components of the Hedgehog signalling pathway, Gli2, Gli3 and X-Smoothened, are expressed in this narrow peripheral epithelium. We show that this zone contains cells that are in the proliferative state. This equivalent region in the adult mammalian eye, the pigmented ciliary epithelium, has been identified as a zone in which retinal stem cells reside. These data, combined with double labelling and the use of other retinal pigment epithelium markers, show that the retinal pigment epithelium of tadpole embryos has a molecularly distinct peripheral to central axis. In addition, Gli2, Gli3 and X-Smoothened are also expressed in the neural retina, in the most peripheral region of the ciliary marginal zone, where retinal stem cells are found in Xenopus, suggesting that they are good markers for retinal stem cells. To test the role of the Hedgehog pathway at different stages of retinogenesis, we activated the pathway by injecting a dominant-negative form of PKA or blocking it by treating embryos with cyclopamine. Embryos injected or treated at early stages display clear proximodistal defects in the retina. Interestingly, the main phenotype of embryos treated with cyclopamine at late stages is a severe defect in RPE differentiation. This study thus provides new insights into the role of Hedgehog signalling in the formation of the proximodistal axis of the eye and the differentiation of retinal pigment epithelium.