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Upstream
Kinds of Upstream Terms modified by Upstream Selected AbstractsReach for the Stars: A Strategic Bidding GameECONOMICA, Issue 272 2001Lynne M. Pepall We examine two,sided competition in a duopoly market for differentiated products. Downstream, the two firms compete in prices. Upstream, they compete in bidding to hire talent input and there is one unique superstar. The outcome depends on the downstream effect of only one firm employing the superstar. When this intensifies downstream competition, both firms are worse off than they would be if no superstar talent were available. When the hiring of the superstar softens downstream competition, both firms benefit, but a ,winner's curse' emerges in which the firm winning the superstar talent earns less profit than its rival. [source] Activation of Epstein-Barr virus/C3d receptor (gp140, CR2, CD21) on human cell surface triggers pp60src and Akt-GSK3 activities upstream and downstream to PI 3-kinase, respectivelyEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2003Monique Barel Abstract We previously demonstrated that CR2 activation on human B lymphocyte surface specifically triggered tyrosine phosphorylation of the 95-kDa nucleolin, this leading to its binding on SH2 domainsof p85 sub-unit of PI 3-kinase and to activation of this enzyme. The specificity of CR2 pathway was clearly demonstrated as neither CD19 nor BCR could induce tyrosine phosphorylation of nucleolin in normal B lymphocytes. These data led us to investigate herein additional molecular events, which were triggered by CR2 activation, upstream and downstream to PI 3-kinase activation. Upstream, we demonstrated that pp60src, a tyrosine kinase of the src family, was involved in tyrosine phosphorylation of nucleolin, while syk tyrosine kinase was not. We also demonstrated a direct protein-proteininteraction of pp60src with nucleolin in a CR2-dependent and CD19-independent pathway. Downstream, we demonstrated that CR2 activation also triggered Akt and GSK3 enzyme activation, this pathway being under the control of pp60src tyrosine kinase activation. These regulatory functions of activated CR2 were specific as independent of syk tyrosine kinase and of CD19 and BCR activation. Thus, CR2 activation recruits a specific mechanism to activate PI 3-kinase and its subsequent pathways, this mechanism being different to those recruited by CD19 and BCR. [source] Characteristics and rehabilitation of the spawning habitats of the sea trout, Salmo trutta, in Gotland (Sweden)FISHERIES MANAGEMENT & ECOLOGY, Issue 1 2004J.-F. Rubin Abstract Characteristics of the natural spawning habitat of sea trout, Salmo trutta L., were studied in Själsöån, a small stream of Gotland, Sweden, from 1992 to 1999. Each year, trout spawned on 17 ± 7 different areas (156 places ha,1). Most of the spawning grounds were used every year. Overcutting was evident for at least 60% of the spawning grounds. Fish spawned on a gravel of 19 ± 7 mm in diameter. From 1978 to 1992, 242 artificial spawning grounds were constructed by the Gotland Sport Fishermen Association in four Gotland streams. A sediment trap was dug upstream to the spawning grounds. The artificial spawning ground comprised of a downstream V-shape stream deflector of large stones with a log weir at the narrowest point of the deflector. Upstream of the dam, 15,60 mm diameter gravel was deposited to constitute the spawning ground substratum. To keep the installation efficient, maintenance is needed every year before the spawning season. [source] Sodium Borohydride Hydrolysis as Hydrogen Generator: Issues, State of the Art and Applicability Upstream from a Fuel CellFUEL CELLS, Issue 3 2010U. B. Demirci Abstract Today there is a consensus regarding the potential of NaBH4 as a good candidate for hydrogen storage and release via hydrolysis reaction, especially for mobile, portable and niche applications. However as gone through in the present paper two main issues, which are the most investigated throughout the open literature, still avoid NaBH4 to be competitive. The first one is water handling. The second one is the catalytic material used to accelerate the hydrolysis reaction. Both issues are objects of great attentions as it can be noticed throughout the open literature. This review presents and discusses the various strategies which were considered until now by many studies to manage water and to improve catalysts performances (reactivity and durability). Published studies show real improvements and much more efforts might lead to significant overhangs. Nevertheless, the results show that we are still far from envisaging short-term commercialisation. [source] Fish assemblage changes relative to environmental factors and time in the Warta River, Poland, and its oxbow lakesJOURNAL OF FISH BIOLOGY, Issue 2 2004T. Penczak Four oxbow lakes and two neighbouring sections of their parent Warta River (Odra River system, Poland) were sampled to investigate differences in fish assemblages between habitats in 1999,2000. Additional comparisons were made with 12 other oxbow lakes in this section of the river that were sampled 30,40 years ago. Downstream of a man-made reservoir, higher species number, diversity and evenness were recorded in oxbow lakes than in the river channel. Upstream of the reservoir, differences in these variables from both habitats were insignificant. Fluvial and stagnant water samples were clearly separated in the multivariate space of a detrended correspondence analysis (DCA). For two oxbow lakes and two neighbouring sections of the Warta River, 12 abiotic and biotic environmental variables were available, and only velocity, water temperature and conductivity were significantly correlated with canonical correspondence analysis (CCA) axes. Fish assemblages of four recently investigated oxbow lakes were clearly separated in the multivariate space of DCA from other neighbouring oxbows sampled 30,40 years ago. Species previously subdominant were becoming rare. Oxbow lakes that are continuously or at least periodically connected with the channel are indispensable for maintaining high biodiversity and a sustainable fishery in the river system. [source] "Thinking Upstream" to Confront PrematurityJOURNAL OF OBSTETRIC, GYNECOLOGIC & NEONATAL NURSING, Issue 5 2003Margaret H. Kearney Associate Editor No abstract is available for this article. [source] The vanG glycopeptide resistance operon from Enterococcus faecalis revisitedMOLECULAR MICROBIOLOGY, Issue 3 2003Florence Depardieu Summary Acquired VanG-type resistance to vancomycin (MIC = 16 µg ml,1) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d -alanine- d -serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3, end encoded VanG, a d -Ala:d -Ser ligase, VanXYG, a putative bifunctional d,d -peptidase and VanTG, a serine racemase: VanG and VanTG were implicated in the synthesis of d -Ala:d -Ser as in VanC- and VanE-type strains. Upstream from the structural genes for these proteins were vanWG with unknown function and vanYG containing a frameshift mutation which resulted in premature termination of the encoded protein and accounted for the lack of UDP-MurNAc-tetrapeptide in the cytoplasm. Without the frameshift mutation, VanYG had homology with Zn2+ dependent d,d -carboxypeptidases. The 5, end of the gene cluster contained three genes vanUG, vanRG and vanSG encoding a putative regulatory system, which were co-transcribed constitutively from the PYG promoter, whereas transcription of vanYG,WG,G,XYG,TG was inducible and initiated from the PYG promoter. Transfer of VanG-type glycopeptide resistance to E. faecalis JH2-2 was associated with the movement, from chromosome to chromosome, of genetic elements of c. 240 kb carrying also ermB -encoded erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donor and transconjugants revealed the same 4 bp direct repeats and 22 bp imperfect inverted repeats that delineated the large element. [source] Swimming Upstream: Collaborative Approaches to Watershed Management Edited by Paul Sabatier, Will Focht, Mark Lubell, Zev Trachtenberg, Arnold Vedlitz and Marty MatlockNATURAL RESOURCES FORUM, Issue 2 2006Randall Hannum No abstract is available for this article. [source] Föhn as a response to changing upstream and downstream air massesTHE QUARTERLY JOURNAL OF THE ROYAL METEOROLOGICAL SOCIETY, Issue 635 2008Georg J. Mayr Abstract Observations of föhn from the field phase of the Mesoscale Alpine Programme (MAP) are used to study how differences between the air masses upstream and downstream of the central Alpine crest determine whether the flow can descend to the lee as either shallow föhn, when it passes through passes in the mountains, or deep föhn, when it overflows the Alpine crest. First, the föhn case of 30 October 1999 is examined using ECMWF analyses and radiosonde data at various upstream and downstream locations. Additional measurements from aircraft, dropsondes, an instrumented car and automatic weather stations are then used for a detailed study of the föhn flow across the Brenner Pass. Advection of cold air around the eastern edges of the Alps and warm air around the western edge of the Alps ahead of a synoptic ridge set up a reservoir of colder air on the south side of the Alps and a reservoir of warmer air to the north. The depth to where the air was colder on the southern side was sufficient for a shallow föhn to flow through the pass. After the passage of the ridge axis, synoptic cold air advection provided another source of colder air, this time from the southwest, growing deeper with time and having a synoptically imposed cross-barrier flow component. The maximum depth to where the air upstream was colder than downstream extended just above the peaks of the highest mountains. An analysis of the detailed measurements across the Brenner Pass showed that this depth was also the top of the layer that descended and accelerated down the lee slopes of the Wipp Valley. Upstream, air above the föhn layer had an even stronger cross-barrier component yet did not descend because it did not have lower potential temperatures than the downstream side at that level. Deep föhn never developed. An examination of other well-documented MAP föhn cases confirmed the conclusion from the 30 October event that shallow and deep föhns , at least for the central Alps , are mostly a response to differences in air masses between the upstream and downstream side. A cross-barrier component of the flow was only a modification but in itself not sufficient to cause the flow to both descend and accelerate down the lee slope, unless potential temperatures on the upstream side were lower in this layer than on the downstream side. Copyright © 2008 Royal Meteorological Society [source] Diagnosing infections,,current and anticipated technologies for point-of-care diagnostics and home-based testingCLINICAL MICROBIOLOGY AND INFECTION, Issue 8 2010L. Bissonnette Clin Microbiol Infect 2010; 16: 1044,1053 Abstract In recent years, we have witnessed many transitions in healthcare systems around the globe. For example, population expansion and ageing, and the human immunodeficiency virus (HIV)-AIDS epidemics, have exerted pressure to decentralize the practice of healthcare outside of traditional settings to bring care to those in need. Upstream of patient management, diagnosis is aimed at adequately orienting medical decisions, and considerable efforts have been made to make this process faster and more efficient. However, there are several diseases and medical conditions that may/will benefit from technologies and tests that can be performed closer to the patient, at the point of care or even in the home. In this review, and in light of the paradox that technology and assay developers and healthcare officials must take into consideration for advancing human health in developed and developing countries, we present an overview of rapid diagnosis of infectious diseases at the point of care and of technologies that may contribute to enhancement of the worldwide point-of-care testing market. [source] Birth defects caused by mutations in human GLI3 and mouse Gli3 genesCONGENITAL ANOMALIES, Issue 1 2010Ichiro Naruse ABSTRACT GLI3 is the gene responsible for Greig cephalopolysyndactyly syndrome (GCPS), Pallister,Hall syndrome (PHS) and Postaxial polydactyly type-A (PAP-A). Genetic polydactyly mice such as Pdn/Pdn (Polydactyly Nagoya), XtH/XtH (Extra toes) and XtJ/XtJ (Extra toes Jackson) are the mouse homolog of GCPS, and Gli3tmlUrtt/Gli3tmlUrt is produced as the mouse homolog of PHS. In the present review, relationships between mutation points of GLI3 and Gli3, and resulting phenotypes in humans and mice are described. It has been confirmed that mutation in the upstream or within the zinc finger domain of the GLI3 gene induces GCPS; that in the post-zinc finger region including the protease cleavage site induces PHS; and that in the downstream of the GLI3 gene induces PAP-A. A mimicking phenomenon was observed in the mouse homolog. Therefore, human GLI3 and mouse Gli3 genes have a common structure, and it is suggested here that mutations in the same functional regions produce similar phenotypes in human and mice. The most important issue might be that GCPS and PHS exhibit an autosomal dominant trait, but mouse homologs, such as Pdn/Pdn, XtH/XtH, XtJ/XtJ and Gli3tmlUrt/Gli3tmlUrt, are autosomal recessive traits in the manifestation of similar phenotypes to human diseases. It is discussed here how the reduced amounts of the GLI3 protein, or truncated mutant GLI3 protein, disrupt development of the limbs, head and face. [source] Phenotypic differences in the brains and limbs of mutant mice caused by differences of Gli3 gene expression levelsCONGENITAL ANOMALIES, Issue 2 2001Ichiro Naruse ABSTRACT, The genetic polydactyly/arhinencephaly mouse, Pdn/Pdn, exhibits severe polydactyly both in the fore-and hindlimbs, agenesis of the olfactory bulbs, corpus callosum, anterior commissure, and hydrocephalus. A candidate gene for the Pdn mouse has been speculated to be Gli3, because Pdn has been considered to be an allele of Xt whose responsible gene has been clarified to be Gli3. Recently, it has been cleared that retro-transposons are inserted into nitron 3, upstream of zinc finger domain, of the Gli3 gene in the Pdn mouse, resulting to the severe suppression of Gli3 gene expression in Pdn/Pdn embryos. Meanwhile, XtJ/XtJ mice exhibit more severe polydactyly than that of Pdn/Pdn. Arhinencephaly and microholoprosencephaly including agenesis of the olfactory bulbs, corpus callosum, anterior commissure, hippocampal commissure, habenular commissure, and posterior commissure, and moreover, the cerebral cortical plates and hippocampus are not formed in the XtJ/XtJ mice. The XtJ/XtJ mouse has a large deletion in Gli3 structural gene and shows null expression. From these corroborations, we speculated that the differences in the Gli3 gene expression levels resulted in the phenotypic differences between the Pdn/Pdn and XtJ/XtJ mice. [source] Effects of Habitat Disturbance on Stream Salamanders: Implications for Buffer Zones and Watershed ManagementCONSERVATION BIOLOGY, Issue 3 2003John D. Willson To minimize the impacts of development on aquatic habitats, numerous conservation measures have been implemented, including the use of riparian buffer zones along streams and rivers. We examined the effectiveness of current buffer-zone systems for management of small watersheds in conserving stream-dwelling salamander populations in 10 small streams ( draining <40.5 ha ) in the western Piedmont of North Carolina. We captured salamanders by means of funnel traps and systematic dipnetting and used a geographic information system to calculate the percentage of disturbed habitat within the watershed of each stream and within 10.7-, 30.5-, and 61.0 -m buffer zones around each stream, upstream from our sampling locations. Although the relative abundance of salamanders was strongly inversely proportional to the percentage of disturbed habitat in the entire watersheds ( R2 = 0.71 for Desmognathus fuscus and 0.48 for Eurycea cirrigera ), we found little to no correlation between the relative abundance of salamanders and the percentage of disturbed habitat present within buffer zones ( R2 = 0.06,0.27 for D. fuscus and 0.01,0.07 for E. cirrigera ). Thus, conservation efforts aimed at preserving salamander populations in headwater streams must consider land use throughout entire watersheds, rather than just preserving small riparian buffer zones. Resumen: La destrucción y degradación del hábitat se encuentra entre la mayores amenazas a la vida silvestre, junto con el aumento global de la población humana. Para minimizar los impactos del desarrollo sobre hábitats acuáticos, se han instrumentado numerosas medidas de conservación, incluyendo el uso de zonas de amortiguamiento riparias a lo largo de arroyos y ríos. Examinamos la efectividad de los actuales sistemas de zonas de amortiguamiento usados en el manejo de cuencas pequeñas para la conservación de poblaciones de salamandras de arroyo en 10 arroyos pequeños ( que drenan <40.5 ha ) al pie de monte del occidente de Carolina del Norte. Capturamos salamandras con trampas de embudo y mediante el uso sistemático de redes y utilizamos un sistema de información geográfica para calcular el porcentaje de hábitat perturbado dentro de la cuenca de cada arroyo y dentro de zonas de amortiguamiento de 10.7-, 30.5- y 61.0-m alrededor de cada arroyo, río arriba de nuestros sitios de muestreo. Aunque los valores de abundancia relativa de salamandras fueron inversamente proporcionales al porcentaje de hábitat perturbado en el total de las cuencas ( R2 = 0.71 para Desmognathus fuscus y 0.48 para Eurycea cirrigera ), encontramos una correlación débil o inexistente entre las abundancias relativas de salamandras y el porcentaje de hábitat perturbado en las zonas de amortiguamiento R2 = 0.06,0.27 para D. fuscus y 0.01,0.07 para E. cirrigera ). Por lo tanto, los esfuerzos de conservación dirigidos a preservar poblaciones de salamandras de arroyos de cabecera deben tomar en cuenta el uso de la tierra en la superficie entera de las cuencas, en lugar de preservar pequeñas zonas de amortiguamiento riparias. [source] Effects of Habitat Fragmentation by Damming on the Persistence of Stream-Dwelling Charr PopulationsCONSERVATION BIOLOGY, Issue 5 2002Kentaro Morita We assessed the effect of habitat fragmentation by damming on the population persistence of a stream-dwelling fish, the white-spotted charr (Salvelinus leucomaenis), in streams of southwestern Hokkaido, Japan. We sampled for charr at 52 dammed-off sites by electrofishing or snorkeling and measured five habitat characteristics: isolation period, watershed area, gradient, elevation, distance from sea. Of the 52 study sites above dams, white-spotted charr were absent at 17 sites and were present at 35 sites. Because the charr occupied all undammed upstream reaches, the damming would cause the absence of charr upstream. Among five habitat characteristics examined, stepwise logistic-regression analysis showed that disappearance was promoted with increasing isolation period, with decreasing watershed area (i.e., habitat size), and with decreasing gradient. The resulting logistic model explained 82.7% of the present white-spotted charr occurrence and forecasted that 12 of 35 extant populations will disappear after 50 years. Our findings imply that extirpation of small, dammed-off populations is inevitable unless efficient fish ladders are installed or dams are removed. Resumen: La construcción de presas tiene serias consecuencias sobre los ecosistemas acuáticos y uno de los mas serios es el "efecto de barrera" ( la prevención de que un organismo migre a través del sistema). Evaluamos los efectos de la fragmentación del hábitat ocasionados por la construcción de presas en la persistencia de un pez morador de arroyos, la trucha de montaña de manchas blancas ( Salvelinus leucomaenis), en arroyos del suroeste de Hokkaido, Japón. Muestreamos en 52 sitios con represas empleando electropesca o buceo con esnórquel y medimos cinco características del hábitat ( periodo de aislamiento, área de la cuenca, gradiente, elevación y distancia al mar). De los 52 sitios de estudio ubicados arriba de las represas las truchas de montaña estuvieron ausentes en 17 sitios y presentes en 35 sitios. Debido a que las truchas de montaña ocupaban todos los rangos sin represas arroyo arriba, la creación de represas pudo causar la ausencia de truchas de montaña arroyo arriba. Dentro de las cinco características del hábitat examinadas, el análisis de regresión logística mostró que la desaparición era promovida por un incremento en el periodo de aislamiento, con una disminución en el área de la cuenca (i.e. tamaño del hábitat) y con una disminución del gradiente. El modelo logístico resultante explicó 82.7% de la presencia de truchas de montaña y pronosticó que 12 de la 35 poblaciones actuales desaparecerían después de 50 años. Nuestros resultados implican que la extirpación es pequeña y que la exclusión por construcción de represas es inevitable a menos que se instalen desvíos para peces o que se remuevan las presas. [source] Released nucleotides amplify the cilium-dependent, flow-induced [Ca2+]i response in MDCK cellsACTA PHYSIOLOGICA, Issue 3 2009H. A. Praetorius Abstract Aim:, Changes in perfusate flow produce increases in [Ca2+]i in renal epithelial cells. Cultured renal epithelia require primary cilia to sense subtle changes in flow. In perfused kidney tubules this flow response is caused by nucleotide signalling via P2Y2 receptors. It is, however, not known whether nucleotides are released by mechanical stress applied to renal primary cilia. Here we investigate whether nucleotides are released during the cilium-dependent flow response and contribute to the flow-induced, cilium-dependent [Ca2+]i signal. Methods:, MDCK cells loaded with Fluo-4-AM were observed at 37 °C in semi-open single or closed-double perfusion chambers. Results:, Our data suggest a purinergic component of the cilium-dependent flow-response: (1) ATP scavengers and P2 receptor antagonists reduced (55%) the cilium-dependent flow-response; (2) ATP added at subthreshold concentration sensitized the renal epithelia to flow changes; (3) increases in fluid flow transiently enhanced the ATP concentration in the superfusate (measured by biosensor-cells). To test if nucleotides were released in sufficient quantities to stimulate renal epithelia we used non-confluent MDCK cells without cilia as reporter cells. We confirmed that non-confluent cells do not respond to changes in fluid flow. Placing confluent, ciliated cells upstream in the in-flow path of the non-confluent cells made them responsive to fluid flow changes. This phenomenon was not observed if either non-confluent or de-ciliated confluent cells were placed upstream. The [Ca2+]i -response in the non-confluent cells with ciliated cells upstream was abolished by apyrase and suramin. Conclusion:, This suggests that subtle flow changes sensed by the primary cilium induces nucleotide release, which amplifies the epithelial [Ca2+]i -response. [source] Comparative Study of Flat and Round Collectors Using a Validated 1D Fluid Probe ModelCONTRIBUTIONS TO PLASMA PHYSICS, Issue 5-6 2006P. Peleman Abstract In the literature two different types of Gundestrup-like probe designs are proposed: design with flat and with round collectors. In this paper we study the influence of different collector shapes of Gundestrup-like probes on the accuracy of the measurement of the parallel and perpendicular flows. A one dimensional fluid probe model is used for deducing both Mach numbers of the unperturbed flow from the probe data. An analytical expression relates the plasma flow to the measured ion saturation currents collected at the upstream and downstream collecting surfaces of the probe. For flat collectors, the analytical model is validated by comparing it to a two dimensional quasi-neutral Particle In Cell (PIC) simulation code. An extension of the theoretical model then allows us to study round collectors. We performed an accuracy study which showed that systematic errors are introduced when round collectors are employed for determination of the perpendicular flow which is systematically overestimated. The error can reach more than 70% when the perpendicular flow increases and when the angle of the collecting surface with respect to the magnetic field (, , 0)is small. The correct analytical expression is applied to experimental data from Gundestrup probe measurements with round collectors on the CASTOR tokamak. The analysis shows that for these measurements the error introduced by using the expression for flat collectors remains negligible, supporting our former use of the model for flat collectors. A new advanced Gundestrup-like probe design and the motivation for the choice of flat collectors are presented. (© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Health Care Supply Chain Design: Toward Linking the Development and Delivery of Care Globally,DECISION SCIENCES, Issue 2 2009Kingshuk K. Sinha ABSTRACT This article is motivated by the gap between the growing demand and available supply of high-quality, cost-effective, and timely health care, a problem faced not only by developing and underdeveloped countries but also by developed countries. The significance of this problem is heightened when the economy is in recession. In an attempt to address the problem, in this article, first, we conceptualize care as a bundle of goods, services, and experiences,including diet and exercise, drugs, devices, invasive procedures, new biologics, travel and lodging, and payment and reimbursement. We then adopt a macro, end-to-end, supply chain,centric view of the health care sector to link the development of care with the delivery of care. This macro, supply chain,centric view sheds light on the interdependencies between key industries from the upstream to the downstream of the health care supply chain. We propose a framework, the 3A-framework, that is founded on three constructs,affordability, access, and awareness,to inform the design of supply chain for the health care sector. We present an illustrative example of the framework toward designing the supply chain for implantable device,based care for cardiovascular diseases in developing countries. Specifically, the framework provides a lens for identifying an integrated system of continuous improvement and innovation initiatives relevant to bridging the gap between the demand and supply for high-quality, cost-effective, and timely care. Finally, we delineate directions of future research that are anchored in and follow from the developments documented in the article. [source] e-Integration in the Supply Chain: Barriers and Performance,DECISION SCIENCES, Issue 4 2002Markham T. Frohlich ABSTRACT Current opinion holds that Internet-based supply chain integration with upstream suppliers and downstream customers (called "e-integration" in this paper) is superior to traditional ways of doing business. This proposition remains untested, however, and similarly we know little about what are the upstream, internal, and downstream barriers to implementing e-integration. This paper empirically addressed these questions using data from a large single nation study, and found (1) a positive link between e-integration and performance, and (2) that internal barriers impeded e-integration more than either upstream supplier barriers or downstream customer barriers. Findings from this study contribute to our theoretical understanding of implementing change in contemporary supply chains, and have important implications for manufacturers interested in improving their supply chain's performance using the Internet. [source] RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008Aya Nishiyama We carried out RNA interference by expressing short hairpin RNA (shRNA) in the Ciona intestinalis embryo. For this purpose, we identified a gene encoding U6 small nuclear RNA (snRNA) in the C. intestinalis genome. The 1-kb sequence upstream of the U6 snRNA gene was sufficient for directing transcription of short RNA as revealed by Northern blot hybridization. An shRNA-expressing plasmid vector was constructed, in which shRNA-encoding oligonucleotides are inserted downstream of the U6 promoter. An shRNA that contained a sequence homologous to the C. intestinalis tyrosinase gene (Ci-tyrosinase) suppressed melanization of pigment cells in the brain of morphologically normal tailbud embryos. An shRNA that perfectly matched the translated sequence of enhanced green fluorescent protein (EGFP) (a mutant type of Aequorea victoria green fluorescent protein) suppressed the expression of the coelectroporated EGFP transgene. These results suggest that the expression of shRNA interferes with functions of both endogenous and exogenous genes. The shRNA-expressing plasmid constructed in the present study provides an easy and inexpensive alternative for the functional analysis of genes in ascidian embryos. [source] Novel genes involved in canonical Wnt/, -catenin signaling pathway in early Ciona intestinalis embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2008Shuichi Wada We report here characterization of five genes for novel components of the canonical Wnt/, -catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss-of-function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci-PGAP1, a Ciona orthologue of human PGAP1, which encodes GPI (glycosylphosphatidylinositol) inositol-deacylase, Ci-ZF278, a gene encoding a C2H2 zinc-finger protein, Ci-C10orf11, a Ciona orthologue of human C10orf11 that encodes a protein with leucine-rich repeats, Ci-Spatial/C4orf17, a single counterpart for two human genes Spatial and C4orf17, and Ci-FLJ10634, a Ciona orthologue of human FLJ10634 that encodes a member of the J-protein family. Knockdown of each of the genes mimicked , -catenin knockdown and resulted in suppression of the expression of , -catenin downstream genes (Ci-FoxD, Ci-Lhx3, Ci-Otx and Ci-Fgf9/16/20) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild-type, of Ci- , -catenin. Dosage-sensitive interactions were found between Ci-,-catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci- , -catenin in the Wnt/, -catenin signaling pathway in early Ciona embryos. [source] Transgenic analysis of the medaka mesp-b enhancer in somitogenesisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2006Harumi Terasaki Somitogenesis is a critical step during the formation of metameric structures in vertebrates. Recent studies in mouse, chick, zebrafish and Xenopus have revealed that several factors, such as T-box genes, Notch/Delta, Wnt, retinoic acid and FGF signaling, are involved in the specification of nascent somites. By interacting with these pathways, the Mesp2-like bHLH transcription factors are transiently expressed in the anterior presomitic mesoderm and play a crucial role in somite formation. The regulatory mechanisms of Mesp2 and its related genes during somitogenesis have been studied in mouse and Xenopus. However, the precise mechanism that regulates the transcriptional activity of Mesp2 has yet to be determined. In our current report, we identify the essential enhancer element of medaka mesp-b, an orthologue of mouse Mesp2, using transgenic techniques and embryo manipulation. Our results demonstrate that a region of approximately 2.8 kb, upstream of the mesp-b gene, is responsible for both the initiation and anterior localization of mesp-b transcription within a somite primordium. Furthermore, putative motifs for both T-box transcription factors and Notch/Delta signaling are present in this enhancer region and are essential for activity. [source] Requirement for ,B1-crystallin promoter of Xenopus laevis in embryonic lens development and lens regenerationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2005Nobuhiko Mizuno Regulation of the lens-specific ,B1-crystallin promoter in Xenopus laevis was investigated using transgenic larvae and tadpoles. Comparison of the promoter sequence with that of chicken ,B1-crystallin gene indicates significant sequence similarity over a span of several hundred base pairs starting from the transcriptional start site. Remarkably, PL-1 and PL-2 sequences identified in the chicken promoter as essential binding sites of MAF, Pax6 and Prox1 transcription factors were conserved. Mutations of X (Xenopus) PL-1 and XPL-2 sequences eliminated the promoter activity, indicating a conserved mechanism regulating ,B1-crystallin promoter among vertebrate species. A stepwise deletion of the promoter sequence starting from 2800 bp indicated that the proximal 260 bp directly upstream of the transcription initiation site is sufficient for eliciting lens-specific expression, but the 150 bp promoter sequence is inactive despite it containing the XPL-1 and XPL-2 sequences, suggesting the presence of an additional and essential regulatory sequence located between ,150 and ,260 bp. Activity of the ,B1-crystallin promoter during lens regeneration from cornea was examined using transgenic tadpoles and found to have the same dependence on promoter regions as in embryonic lens development, indicating that gene regulation is largely shared by the two lens-generating processes. [source] Functional retinoid receptors in budding ascidiansDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2000Mika Kamimura A homolog of retinoid X receptors (RXR), named PmRXR, was cloned from the budding ascidian, Polyandrocarpa misakiensis. Gel-shift assays revealed that PmRXR and a previously identified P. misakiensis retinoic acid receptor (PmRAR) formed a complex to bind vertebrate-type retinoic acid response element (RARE). Transfection assays were carried out using a reporter gene containing a RARE upstream of lacZ. Two chimeric effector genes were constructed by placing PmRXR and PmRAR cDNA fragments (containing the DNA-binding, ligand-binding and ligand-dependent transactivation domains) downstream of the human RXR, and RAR, cDNA (covering the N-terminal coding region), respectively. Each chimeric cDNA was ligated to a notochord-specific enhancer. In case the embryos were transfected with all three transgenes and treated with retinoic acid (RA), the reporter gene was activated in the notochord cells. The result suggests that the PmRXR/PmRAR complex functions as an RA-dependent transcriptional activator. The PmRXR mRNA was detected in a mesenchymal cell type, called glomerulocyte, in the developing Polyandrocarpa bud. As this cell type has been shown to express PmRAR mRNA, it seems possible that the PmRXR/PmRAR complex mediates RA signaling in this cell type to induce the expression of genes involved in the morphogenesis of the developing bud. [source] Real-time observation of Wnt ,-catenin signaling in the chick embryoDEVELOPMENTAL DYNAMICS, Issue 1 2010Anne C. Rios Abstract A critical mediator of cell,cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Reporter constructs containing multimerized TCF DNA binding sites have been used to detect Wnt ,-catenin dependent activity during animal development. In this report, we have constructed and compared several TCF green fluorescent protein (GFP) reporter constructs. They contained 3, 8, or 12 TCF binding sites upstream of a minimal promoter driving native or destabilized enhanced GFP (EGFP). We have used the electroporation of somites in the chick embryo as a paradigm to test them in vivo. We have verified that they all respond to Wnt signaling in vivo. We have then assessed their efficiency at reflecting the activity of the Wnt pathway. Using destabilized EGFP reporter constructs, we show that somite cells dynamically regulate Wnt/,-catenin,dependent signaling, a finding that was confirmed by performing time-lapse video confocal observation of electroporated embryos. Developmental Dynamics 239:346,353, 2010. © 2009 Wiley-Liss, Inc. [source] Neural protein Olig2 acts upstream of the transcriptional regulator sim1 to specify diencephalic dopaminergic neuronsDEVELOPMENTAL DYNAMICS, Issue 4 2009Nataliya Borodovsky Abstract Neural factors are expressed in neural progenitors and regulate neurogenesis and gliogenesis. Recent studies suggested that these factors are also involved in determining specific neuronal fates by regulating the expression of their target genes, thereby creating transcriptional codes for neuronal subtype specification. In the present study, we show that in the zebrafish the neural gene Olig2 and the transcriptional regulator Sim1 are co-expressed in a subset of diencephalic progenitors destined towards the dopaminergic (DA) neuronal fate. While sim1 mRNA is also detected in mature DA neurons, the expression of olig2 is extinguished prior to terminal DA differentiation. Loss of function of either Olig2 or Sim1 leads to impaired DA development. Finally, Olig2 regulates the expression of Sim1 and gain of function of Sim1 rescues the deficits in DA differentiation caused by targeted knockdown of Olig2. Our findings demonstrate for the first time that commitment of basal diencephalic DA neurons is regulated by the combined action of the neural protein Olig2 and its downstream neuronal specific effector Sim1. Developmental Dynamics 238:826,834, 2009. © 2009 Wiley-Liss, Inc. [source] Transcriptional control of Rohon-Beard sensory neuron development at the neural plate borderDEVELOPMENTAL DYNAMICS, Issue 4 2009Christy Cortez Rossi Abstract Rohon-Beard (RB) mechanosensory neurons are among the first sensory neurons to develop, and the process by which they adopt their fate is not completely understood. RBs form at the neural plate border (NPB), the junction between neural and epidermal ectoderm, and require the transcription factor prdm1a. Here, we show that prior to RB differentiation, prdm1a overlaps extensively with the epidermal marker dlx3b but shows little overlap with the neuroectodermal markers sox3 and sox19a. Birthdating analysis reveals that the majority of RBs are born during gastrulation in zebrafish, suggesting that it is during this period that RBs become specified. Expression analysis in prdm1a and neurogenin1 mutant and dlx3b/dlx4b morpholino-injected embryos suggests that prdm1a is upstream of dlx3b, dlx4b, and neurogenin1 at the NPB. mRNA for neurogenin1 or dlx3b/dlx4b can rescue the lack of RBs in prdm1a mutants. Based on these data, we suggest a preliminary gene regulatory network for RB development. Developmental Dynamics 238:931,943, 2009. © 2009 Wiley-Liss, Inc. [source] Lineage-independent mosaic expression and regulation of the Ciona multidom gene in the ancestral notochordDEVELOPMENTAL DYNAMICS, Issue 7 2007Izumi Oda-Ishii Abstract The transcription factor Ciona Brachyury (Ci-Bra) plays an essential role in notochord development in the ascidian Ciona intestinalis. We characterized a putative Ci-Bra target gene, which we named Ci - multidom, and analyzed in detail its expression pattern in normal embryos and in embryos where Ci - Bra was misexpressed. Ci - multidom encodes a novel protein, which contains eight CCP domains and a partial VWFA domain. We show that an EGFP-multidom fusion protein localizes preferentially to the endoplasmic reticulum (ER), and is excluded from the nucleus. In situ hybridization experiments demonstrate that Ci - multidom is expressed in the notochord and in the anterior neural boundary (ANB). We found that the expression in the ANB is fully recapitulated by an enhancer element located upstream of Ci - multidom. By means of misexpression experiments, we provide evidence that Ci-Bra controls transcription of Ci - multidom in the notochord; however, while Ci-Bra is homogeneously expressed throughout this structure, Ci - multidom is transcribed at detectable levels only in a random subset of notochord cells. The number of notochord cells expressing Ci - multidom varies among different embryos and is independent of developmental stage, lineage, and position along the anterior,posterior axis. These results suggest that despite its morphological simplicity and invariant cell-lineage, the ancestral notochord is a mosaic of cells in which the gene cascade downstream of Brachyury is differentially modulated. Developmental Dynamics 236:1806,1819, 2007. © 2007 Wiley-Liss, Inc. [source] Comparative analysis of Gata3 and Gata2 expression during chicken inner ear developmentDEVELOPMENTAL DYNAMICS, Issue 1 2007Kersti Lilleväli Abstract The inner ear is a complex sensory organ with hearing and balance functions. Gata3 and Gata2 are expressed in the inner ear, and to gain more insight into their roles in otic development, we made a detailed expression analysis in chicken embryos. At early stages, their expression was highly overlapping. At later stages, Gata2 expression became prominent in vestibular and cochlear nonsensory epithelia. In contrast to Gata2, Gata3 was mainly expressed in the developing sensory epithelia, reflecting the importance of this factor in the sensory,neural development of the inner ear. While the later expression patterns of both Gata3 and Gata2 were highly conserved between chicken and mouse, important differences were observed especially with Gata3 during early otic development, providing indications of divergent molecular control during placode invagination in mice and chickens. We also found indications that the regulatory hierarchy observed in mouse, where Gata3 is upstream of Gata2 and Fgf10, could be conserved in chicken. Developmental Dynamics 236:306,313, 2007. © 2006 Wiley-Liss, Inc. [source] Developmental cell death during Xenopus metamorphosis involves BID cleavage and caspase 2 and 8 activationDEVELOPMENTAL DYNAMICS, Issue 8 2006D. Du Pasquier Abstract Elimination of tadpole organs during Xenopus metamorphosis is largely achieved through apoptosis, and recent evidence suggest involvement of the mitochondrial death route and bax-initiated caspase-3 and -9 deployment. However, events upstream of the activation of Bax are unknown. In other models, proteins of the BH3-only group such as BID are known to assure this function. We show that Xenopus bid transcript levels increase at metamorphosis in larval cells destined to disappear. This increase correlates with an abrupt rise in Caspase-2 and -8 mRNA levels and an enhanced activity of Caspase-2 and -8. In BIDGFP transgenic animal's tail regression is accelerated. The cleavage of BIDGFP fusion protein during natural or T3 -induced metamorphosis was specifically inhibited by caspase-8 inhibitors. Our results show that tail regression at metamorphosis implicates an apoptotic pathway inducible by T3 hormone in an organ autonomous manner and involving the cell death executioners BID and Caspases-2 and -8. Developmental Dynamics 235:2083,2094, 2006. © 2006 Wiley-Liss, Inc. [source] Xnr2 and Xnr5 unprocessed proteins inhibit Wnt signaling upstream of dishevelledDEVELOPMENTAL DYNAMICS, Issue 4 2005Yasuko Onuma Abstract Nodal and Nodal-related proteins activate the Activin-like signal pathway and play a key role in the formation of mesoderm and endoderm in vertebrate development. Recent studies have shown additional activities of Nodal-related proteins apart from the canonical Activin-like signal pathway. Here we report a novel function of Nodal-related proteins using cleavage mutants of Xenopus nodal-related genes (cmXnr2 and cmXnr5), which are known to be dominant-negative inhibitors of nodal family signaling. cmXnr2 and cmXnr5 inhibited both BMP signaling and Wnt signaling without activating the Activin-like signal in animal cap assays. Pro region construct of Xnr2 and Xnr5 did not inhibit Xwnt8, and pro/mature region chimera mutant cmActivin - Xnr2 and cmActivin- Xnr5 also did not inhibit Xwnt8 activity. These results indicate that the pro domains of Xnr2 and Xnr5 are necessary, but not sufficient, for Wnt inhibition, by Xnr family proteins. In addition, Western blot analysis and immunohistochemistry analysis revealed that the unprocessed Xnr5 protein is stably produced and secreted as effectively as mature Xnr5 protein, and that the unprocessed Xnr5 protein diffused in the extracellular space. These results suggest that unprocessed Xnr2 and Xnr5 proteins may be involved in inhibiting both BMP and Wnt signaling and are able to be secreted to act on somewhat distant target cells, if these are highly produced. Developmental Dynamics 234:900,910, 2005. © 2005 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||||||||||||||