pinecone/core-2020-05-10-deduplication huggingface dataset:
High-Frequency Generation of Transgenic Zebrafish Which Reliably Express GFP in Whole Muscles or the Whole Body by Using Promoters of Zebrafish Origin

AbstractDespite a number of reports on transgenic zebrafish, there have been no reports on transgenic zebrafish in which the gene is under the control of a promoter of zebrafish origin. Neither have there been reports on transgenic zebrafish in which the gene is under the control of a tissue-specific promoter/enhancer. To investigate whether it is possible to generate transgenic zebrafish which reliably express a reporter gene in specific tissues, we have isolated a zebrafish muscle-specificactin(α-actin) promoter and generated transgenic zebrafish in which the green fluorescent protein (GFP) reporter gene was driven by this promoter. In total, 41 GFP-expressing transgenic lines were generated with a frequency of as high as 21% (41 of 194), and GFP was specifically expressed throughout muscle cells in virtually all of the lines (40 of 41). Nonexpressing transgenic lines were rare. This demonstrates that a tissue-specific promoter can reliably drive reporter gene expression in transgenic zebrafish in a manner identical to the control of the endogeneous expression of the gene. Levels of GFP expression varied greatly from line to line; i.e., fluorescence was very weak in some lines, while it was extremely high in others. We also isolated a zebrafish cytoskeletal β-actinpromoter and generated transgenic zebrafish using a β-actin–GFP construct. In all of the four lines generated, GFP was expressed throughout the body like the β-actingene, demonstrating that consistent expression could also be achieved in this case. In the present study, we also examined the effects of factors which potentially affect the transgenic frequency or expression levels. The following results were obtained: (i) expression levels of GFP in the injected embryo were not strongly correlated to transgenic frequency; (ii) the effect of the NLS peptide (SV40 T antigen nuclear localization sequence), which has been suggested to facilitate the transfer of a transgene into embryonic nuclei, remained to be elusive; (iii) a plasmid vector sequence placed upstream of the construct might reduce the expression levels of the reporter gene
Autonomous Neural Axis Formation by Ectopic Expression of the Protooncogene c-ski

AbstractTheskioncogene was originally isolated as an avian retroviral gene with the ability to induce quail embryonic cells to differentiate into muscle. Mice containing a chicken c-skitransgene exhibit postnatal hypertrophy of skeletal muscle.Xenopusski (Xski) protein is maternal and present throughout early development. We show that overexpression ofXskiRNA inXenopusembryos results in the cell-autonomous induction of secondary neural axis formation. Injection ofXskiRNA into prospective endodermal cells resulted in the formation of an ectopic neural tube-like structure and cells derived from the injected blastomeres populated the spinal cord. InjectedXskiRNA was able to induce neural-specific gene expression directly in ectodermal explants in the absence of the expression of mesodermal markers. The widespread distribution of ski protein in the early gastrula embryo including the dorsal animal region supports a role forskiin neural axis formationin vivo
The Role of Intracellular Alkalinization in the Establishment of Anterior Neural Fate inXenopus

AbstractOur previous work demonstrated thatXenopusectoderm cells undergo an alkalinization in response to planar inductive signals during neural induction in explants. We have examined the role of intracellular alkalinization in the establishment of anterior neural fate. First, RT-PCR was used to examine neural-specific gene expression in planar explants in which the alkalinization is prevented by treatment with 4,4′-dihydrodiisothiocyanatostilbene-2,2′-disulfonate (H2DIDS). In explants cultured in the presence of H2DIDS, expression ofNCAMand the anterior neural geneotx2is greatly reduced or absent. Second, neural-specific gene expression was examined in isolates of uninduced animal cap ectoderm cultured in the presence of either methylamine or ammonium chloride.NCAM, otx2,and the anterior neural inducernogginwere expressed in alkalinized ectoderm, while the more posterior neural markerskrox-20andHox B9were undetectable. Expression ofNCAM, otx2,andnogginwas observed at stage 11 in both alkalinized ectoderm and the newly induced neural plate, suggesting that intracellular alkalinization could contribute to propagation of noggin signaling through the dorsal ectoderm. Alkalinization of uninduced ectoderm at stage 10.5 led to an upregulation ofotx2within 15 min. Activation ofNCAMexpression in alkalinized dissociated cells was identical to that observed in intact animal caps, indicating that alkalinization-mediated changes in gene expression do not require cell–cell contact. Finally, the effects of intracellular alkalinization on protein tyrosine phosphorylation were investigated using 2D gel electrophoresis and immunoblots probed with an anti-phosphotyrosine antibody. Several phosphorylated proteins detected in induced and alkalinized ectoderm were greatly reduced or absent in uninduced ectoderm, indicating that alkalinization elicits alterations in tyrosine phosphorylation similar to some of those observed during neural inductionin vivo.Our results indicate that intracellular alkalinization plays a critical role in the activation of anterior neural-specific gene expression and that alkalinization may act by regulating the activity of a tyrosine kinase or phosphatase
Symmetry Breaking inDictyosteliumMorphogenesis: Evidence That a Combination of Cell Cycle Stage and Positional Information Dictates Cell Fate

AbstractThe event that visibly breaks the symmetry of theDictyosteliumaggregate is the formation of a nipple-shaped tip at its apex, but there has been considerable debate as to the equivalence of cells entering development, particularly with regard to the effect of cell cycle position at the onset of starvation. We show that there is a strong correlation between cell cycle position at the time of starvation and the subsequent expression of a marker of tip cell differentiation. Cells starved in late-G2 phase selectively differentiate into tip cells. Previous evidence indicated that tip cells differentiate at the extreme periphery of the aggregate and then move to the apex. Taken in combination, these data suggest that cell cycle position at the onset of starvation affects differentiation fate by determining a cell's probable position within the aggregate, i.e., that both cell cycle and positional information are used to generate pattern
Translational Activation and Cytoplasmic Polyadenylation of FGF Receptor-1 Are Independently Regulated duringXenopusOocyte Maturation

AbstractFGF signaling is critical for establishing theXenopus laevisembryonic body plan and requires the expression of functional FGF receptor during early embryogenesis. FGF receptor-1 (XFGFR) maternal mRNA is present in immature oocytes, but the protein is not expressed until oocyte maturation. In this report we demonstrate that endogenous XFGFR translation begins just prior to germinal vesicle breakdown and that translation depends on completion of earlier meiotic events. We show that the previously identified XFGFR 3′UTR translation inhibitory element (TIE), which is necessary and sufficient for repressing translation in the immature oocyte, also regulates the onset of translation during oocyte maturation. In addition we demonstrate that cytoplasmic polyadenylation of XFGFR RNA is regulated independently of TIE-mediated translation and is not sufficient to activate the translation of XFGFR. These experiments reveal that polyadenylation and translational activation are separable events in this mRNA, each of which is timed and regulated independently
Expression and Tyrosine Phosphorylation of Eph Receptors Suggest Multiple Mechanisms in Patterning of the Visual System

AbstractThe EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have usedin situhybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells. In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization
No turning,a Mouse Mutation Causing Left–Right and Axial Patterning Defects

AbstractPatterning along the left/right axes helps establish the orientation of visceral organ asymmetries, a process which is of fundamental importance to the viability of an organism. A linkage between left/right and axial patterning is indicated by the finding that a number of genes involved in left/right patterning also play a role in anteroposterior and dorsoventral patterning. We have recovered a spontaneous mouse mutation causing left/right patterning defects together with defects in anteroposterior and dorsoventral patterning. This mutation is recessive lethal and was namedno turning (nt)because the mutant embryos fail to undergo embryonic turning.ntembryos exhibit cranial neural tube closure defects and malformed somites and are caudally truncated. Development of the heart arrests at the looped heart tube stage, with cardiovascular defects indicated by ballooning of the pericardial sac and the pooling of blood in various regions of the embryo. Interestingly, inntembryos, the direction of heart looping was randomized.Nodalandlefty,two genes that are normally expressed only in the left lateral plate mesoderm, show expression in the right and left lateral plate mesoderm.Lefty,which is normally also expressed in the floorplate, is not found in the prospective floorplate ofntembryos. This suggests the possibility of notochordal defects. This was confirmed by histological analysis and the examination ofsonic hedgehog, Brachyury,andHNF-3βgene expression. These studies showed that the notochord is present in the earlyntembryo, but degenerates as development progresses. Overall, these findings support the hypothesis that the notochord plays an active role in left/right patterning. Our results suggest thatntmay participate in this process by modulating the notochordal expression ofHNF-3β
Differential Regulation ofchordinExpression Domains in Mutant Zebrafish

AbstractPatterning along the dorsal–ventral (D–V) axis ofXenopusandDrosophilaembryos is believed to occur through a conserved molecular mechanism, with homologous proteins Chordin and Short gastrulation (Sog) antagonizing signaling by bone morphogenetic protein 4 (BMP-4) and Decapentaplegic (Dpp), respectively. We have isolated a zebrafish gene that is highly homologous tochordinandsogwithin cysteine-rich domains and exhibits conserved aspects of expression and function. As inXenopusembryos, zebrafishchordinis expressed in the organizer region and transiently in axial mesoderm. Injection of zebrafishchordinmRNA to the ventral side ofXenopusembryos induced secondary axes. Ectopic overexpression in zebrafish resulted in an expansion of paraxial mesoderm and neurectoderm at the expense of more lateral and ventral derivatives, producing a range of defects similar to those of dorsalized zebrafish mutants (Mullinset al.,1996). In accordance with the proposed function ofchordinin D–V patterning, dorsalized zebrafish mutants showed expanded domains ofchordinexpression by midgastrulation, while some ventralized mutants had reduced expression; however, in all mutants examined, early organizer expression was unaltered. In contrast toXenopus,zebrafishchordinis also expressed in paraxial mesoderm and ectoderm and in localized regions of the developing brain, suggesting that there are additional roles forchordinin zebrafish embryonic development. Surprisingly, paraxial mesodermal expression ofchordinappeared unaltered inspadetailmutants that later lack trunk muscle (Kimmelet al.,1989), while axial mesodermal expression was affected. This finding reveals an unexpected function forspadetailin midline mesoderm and in differential regulation ofchordinexpression during gastrulation
Suppressors of Ectopic UNC-5 Growth Cone Steering Identify Eight Genes Involved in Axon Guidance inCaenorhabditis elegans

AbstractThe UNC-5 guidance receptor, in response to the UNC-6/netrin path cue, orients growing axons in a dorsal direction along the epidermis ofCaenorhabditis elegans.When ectopically expressed in the touch neurons, which normally extend ventrally or longitudinally, UNC-5 is able to reorient their axons toward the dorsal side in an UNC-6-dependent manner. This forms the basis of a genetic screen to identify other mutations that, likeunc-6mutations, suppressunc-5-induced growth cone guidance. These mutations may identify new components required for pioneer axon guidance byunc-5.In this paper, we describe eight genes that are required for ectopicunc-5-induced growth cone steering. Mutations in four of these identify the previously known axon guidance genesunc-6, unc-40, unc-34,andunc-44and mutations in four others identify the novel genesunc-129, seu-1, seu-2,andseu-3.Several of these mutations cause axon guidance defects similar to those found inunc-5mutants. We propose that some or all of these genes may function in a developmentally importantunc-5signaling pathway
Phosphorylation of Protein Kinase C-Related Kinase PRK2 during Meiotic Maturation of Starfish Oocytes

AbstractThe resumption of meiosis in the developing starfish oocyte is the result of intracellular signaling events initiated by 1-methyladenine stimulation. One of the earliest detectable kinase activities during meiotic maturation of starfish oocytes is a protein kinase C or PKC-like activity. In this study, several isoforms of protein kinase C were cloned from the oocyte; however, the most abundant PKC-like maternal transcript corresponds to protein kinase C-related kinase 2 (PRK2). PRK2 is expressed in the immature oocyte and at least until germinal vesicle breakdown. Subcellular localization of PRK2 revealed a cytoplasmic distribution in the immature oocyte, which, during meiotic maturation, remained in the cytoplasm but also localized to the disintegrating germinal vesicle. Significantly, PRK2 is phosphorylatedin vivoin response to 1-methyladenine which precedes MPF activation, making PRK2 a candidate regulator of early signaling events of meiotic maturation
The Calcium Transient in Sea Urchin Eggs during Fertilization Requires the Production of Inositol 1,4,5-Trisphosphate

AbstractThe production of inositol 1,4,5-trisphosphate (InsP3) has been reported to mediate the transient rise in intracellular Ca2+activity ([Ca2+]i) in sea urchin eggs during fertilization. However, direct evidence of an absolute requirement for generation of InsP3during fertilization is still lacking. We investigated this question by blocking the InsP3synthesizing enzyme phospholipase C (PLC) during fertilization with U73122, an aminosteroid. U73122 inhibited the sperm-induced Ca2+release in a dose-dependent manner, although above 15 μM U73122 eggs showed an elevated resting [Ca2+]iand a lower fertilization rate. The inhibition of Ca2+transient by U73122 was not due to a failure of fertilization, since incorporated sperm nuclei were evident in eggs used to measure the Ca2+response. U73122 also prevented the accompanying rise in intracellular pH (pHi), which is mediated by the activation of the Na+-H+antiporter. The antiporter is regulated through activation of protein kinase C by 1,2-diacylglycerol, which is the other hydrolytic product of phosphatidylinositol 4,5-bisphosphate by PLC. Further evidence of the specificity of U73122 action was inhibition of the increase in InsP3mass during the first 2 min of fertilization. In addition, U73122 inhibited the GTPγS-induced Ca2+release and pHirise in unfertilized eggs. These results suggested that the transient rise in Ca2+in sea urchin during fertilization requires the production of InsP3
A Molecular Analysis of Hyalin—A Substrate for Cell Adhesion in the Hyaline Layer of the Sea Urchin Embryo

AbstractThe hyaline layer of echinoderm embryos is an extraembryonic matrix that functions as a substrate for cell adhesion through early development. The major constituent of the hyaline layer is the protein hyalin, a fibrillar glycoprotein of approximately 330 kDa that multimerizes in the presence of calcium. Here we provide a molecular characterization of hyalin and identify a region of the protein that is important for its function in cell adhesion. Partial hyalin cDNAs were identified from two sea urchin species,Strongylocentrotus purpuratusandLytechinus variegatus,by screening expression libraries with monoclonal antibodies to hyalin. The cDNAs each encode a tandemly arranged series of conserved repeats averaging 84 amino acids. These hyalin repeats are as similar between the two species as they are to repeats within each species, suggesting a strong functional conservation. Analysis of this repeat shows that it is a unique sequence within the GenBank database with only weak similarity to mucoid protein sequences. The hyalin mRNA is approximately 12 kb in length and is present in developing oocytes coincident with the appearance of cortical granules, the vesicle in which the hyalin protein is specifically packaged. The mRNA is present throughout oogenesis but is rapidly lost at oocyte maturation so that eggs and early embryos have no detectable hyalin mRNA. The hyalin protein, however, remains at relatively constant levels throughout development. Thus, all the hyalin protein present during early development, when no RNA is detectable, is maternally derived and exocytosed from cortical granules at fertilization. Hyalin mRNA reaccumulates in embryos beginning at the mesenchyme blastula stage; a RNA gel blot andin situhybridization analysis of gastrulae and larvae shows a progressive confinement of hyalin mRNA to the aboral ectoderm. Recombinant hyalin containing the tandem repeat region of the protein was expressed in bacteria and is shown to serve as an adhesive substrate, almost equal to that of native hyalin, in cell adhesion assays. This adhesive activity is partially blocked by dilute hyalin monoclonal antibody Tg-HYL to the same extent as that for native hyalin. Thus, this hyalin repeat region appears to contain the ligand for the hyalin cell surface receptor. These data help explain some of the classic functions ascribed to the hyalin protein in early development and now enable investigators to focus on the mechanisms of cell interactions with the hyaline layer
Pax2 Expression and Retinal Morphogenesis in the Normal andKrdMouse

AbstractTheKidney and retinal defects (Krd)mouse carries a 7-cM transgene-induced deletion on chromosome 19 that includes thePax2locus. Adult mice heterozygous for theKrddeletion (Krd/+) are haploid forPax2and have a variable, semidominant phenotype characterized by structural defects of the kidney, retina, and optic disc. Renal and ocular anomalies present in heterozygousPax2mutants in both mice and humans support the hypothesis that haploinsufficiency ofPax2underlies theKrdphenotype. To understand the embryonic basis of ocular defects observed in adultKrd/+ mice, we used immunohistochemistry, digital three-dimensional reconstructions, and quantitative morphometry to examine Pax2 protein distribution and ocular development in normal andKrd/+ mice from E10.5 to P2. In +/+ embryos, Pax2 immunopositive (Pax2+) cells demarcate the embryonic fissure as it forms in the ventral optic cup and optic stalk. After closure of the embryonic fissure, Pax2 immunostaining disappears from the ventral retina, but persists in a cuff of cells encircling the developing optic disc, the site where ganglion cell axons exit the retina. InKrd/+ embryos, Pax2+ cells in the posterior optic cup and the optic stalk undergo abnormal morphogenetic movements and the embryonic fissure fails to form normally. This results in an abnormal organization of the Pax2+ cells and ganglion cell axons at the nascent optic disc. The abnormal morphogenetic movements of the Pax2+ cells in the embryonic retina and optic stalk and the initial misrouting of the ganglion cell axons give rise to retinal and optic disc defects observed in the adultKrd/+ mice
Paraxial-Fated Mesoderm Is Required for Neural Crest Induction inXenopusEmbryos

AbstractNeural crest induction is thought to occur by a two-step process. Axially fated mesoderm induces neural plate, which is then recruited to neural crest by nonneural epidermal ectoderm at the neural plate border. This model suggests a rather indirect role for mesoderm in inducing neural crest. We extensively examined the role of mesoderm in neural crest induction by determining which types of mesoderm induce neural crest cells inXenopusembryos. We found that noggin-dorsalized ventral marginal zone (VMZ) explants differentiate as melanocytes in the absence of axial mesoderm. Dorsalized VMZ is also a potent inducer of melanocytes when juxtaposed to animal cap ectoderm in recombinant explants. Dorsalized VMZ is analogous to the dorsal–lateral marginal zone (DLMZ) region of the embryo. Neural crest-inducing activities of gastrula stage DLMZ and dorsal marginal zone (DMZ) were also compared in recombinant explants. DLMZ was a stronger inducer of neural crest than was DMZ; DLMZ induced high levels of XSlug expression and melanocyte formation in recombinants, whereas DMZ weakly induced neural crest. In whole embryos lacking DLMZ, XSlug expression and melanocyte formation were significantly reduced; in contrast, no significant reduction of XSlug expression or melanocyte formation was seen in embryos lacking a DMZ. These results suggest that paraxial-fated mesoderm plays a central role in neural crest formation by inducing a novel type of lateral neural plate. This lateral neural plate is then recruited to neural crest by adjacent nonneural epidermal ectoderm