62 results on '"Daar IO"'
Search Results
2. Polyethyleneimine/polyethylene glycol-conjugated gold nanoparticles as nanoscale positive/negative controls in nanotoxicology: testing in frog embryo teratogenesis assay- Xenopus and mammalian tissue culture system.
- Author
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Hwang YS, So D, Lee M, Yoon J, Reipa V, Tona A, Yi F, Nelson BC, LaVan DA, Hackley VA, Daar IO, and Cho TJ
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- Animals, Gold toxicity, Polyethyleneimine toxicity, Polyethylene Glycols toxicity, Xenopus laevis, Embryo, Nonmammalian, Teratogens toxicity, Mammals, Teratogenesis, Metal Nanoparticles toxicity
- Abstract
Despite the great potential of using positively charged gold nanoparticles (AuNPs) in nanomedicine, no systematic studies have been reported on their synthesis optimization or colloidal stability under physiological conditions until a group at the National Institute of Standards and Technology recently succeeded in producing remarkably stable polyethyleneimine (PEI)-coated AuNPs (Au-PEI). This improved version of Au-PEI (Au-PEI25kB) has increased the demand for toxicity and teratogenicity information for applications in nanomedicine and nanotoxicology. In vitro assays for Au-PEI25kB in various cell lines showed substantial active cytotoxicity. For advanced toxicity research, the frog embryo teratogenesis assay- Xenopus (FETAX) method was employed in this study. We observed that positively-charged Au-PEI25kB exhibited significant toxicity and teratogenicity, whereas polyethylene glycol conjugated AuNPs (Au-PEG) used as comparable negative controls did not. There is a characteristic avidity of Au-PEI25kB for the jelly coat, the chorionic envelope (also known as vitelline membrane) and the cytoplasmic membrane, as well as a barrier effect of the chorionic envelope observed with Au-PEG. To circumvent these characteristics, an injection-mediated FETAX approach was utilized. Like treatment with the FETAX method, the injection of Au-PEI25kB severely impaired embryo development. Notably, the survival/concentration curve that was steep when the standard FETAX approach was employed became gradual in the injection-mediated FETAX. These results suggest that Au-PEI25kB may be a good candidate as a nanoscale positive control material for nanoparticle analysis in toxicology and teratology.
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- 2023
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3. Wnt4 and ephrinB2 instruct apical constriction via Dishevelled and non-canonical signaling.
- Author
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Yoon J, Sun J, Lee M, Hwang YS, and Daar IO
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- Constriction, Morphogenesis physiology, Neural Tube, Ephrin-B2 genetics, Signal Transduction physiology
- Abstract
Apical constriction is a cell shape change critical to vertebrate neural tube closure, and the contractile force required for this process is generated by actin-myosin networks. The signaling cue that instructs this process has remained elusive. Here, we identify Wnt4 and the transmembrane ephrinB2 protein as playing an instructive role in neural tube closure as members of a signaling complex we termed WERDS (Wnt4, EphrinB2, Ror2, Dishevelled (Dsh2), and Shroom3). Disruption of function or interaction among members of the WERDS complex results in defects of apical constriction and neural tube closure. The mechanism of action involves an interaction of ephrinB2 with the Dsh2 scaffold protein that enhances the formation of the WERDS complex, which in turn, activates Rho-associated kinase to induce apical constriction. Moreover, the ephrinB2/Dsh2 interaction promotes non-canonical Wnt signaling and shows how cross-talk between two major signal transduction pathways, Eph/ephrin and Wnt, coordinate morphogenesis of the neural tube., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)
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- 2023
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4. Male infertility-associated Ccdc108 regulates multiciliogenesis via the intraflagellar transport machinery.
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Zhao H, Sun J, Insinna C, Lu Q, Wang Z, Nagashima K, Stauffer J, Andresson T, Specht S, Perera S, Daar IO, and Westlake CJ
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- Animals, Centrioles metabolism, Cilia metabolism, Cytoskeletal Proteins metabolism, Humans, Male, Xenopus laevis, Basal Bodies metabolism, Infertility, Male genetics, Membrane Proteins genetics, RNA-Binding Proteins genetics
- Abstract
Motile cilia on the cell surface generate movement and directional fluid flow that is crucial for various biological processes. Dysfunction of these cilia causes human diseases such as sinopulmonary disease and infertility. Here, we show that Ccdc108, a protein linked to male infertility, has an evolutionarily conserved requirement in motile multiciliation. Using Xenopus laevis embryos, Ccdc108 is shown to be required for the migration and docking of basal bodies to the apical membrane in epidermal multiciliated cells (MCCs). We demonstrate that Ccdc108 interacts with the IFT-B complex, and the ciliation requirement for Ift74 overlaps with Ccdc108 in MCCs. Both Ccdc108 and IFT-B proteins localize to migrating centrioles, basal bodies, and cilia in MCCs. Importantly, Ccdc108 governs the centriolar recruitment of IFT while IFT licenses the targeting of Ccdc108 to the cilium. Moreover, Ccdc108 is required for the centriolar recruitment of Drg1 and activated RhoA, factors that help establish the apical actin network in MCCs. Together, our studies indicate that Ccdc108 and IFT-B complex components cooperate in multiciliogenesis., (Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Published under the terms of the CC BY NC ND 4.0 license.)
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- 2022
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5. Zic5 stabilizes Gli3 via a non-transcriptional mechanism during retinal development.
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Sun J, Yoon J, Lee M, Lee HK, Hwang YS, and Daar IO
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- Animals, Cell Differentiation physiology, Gene Expression Regulation, Developmental physiology, Hedgehog Proteins metabolism, Kruppel-Like Transcription Factors metabolism, Repressor Proteins metabolism, Signal Transduction physiology, Trans-Activators metabolism, Transcription Factors metabolism, Xenopus, DNA-Binding Proteins metabolism, Nerve Tissue Proteins metabolism, Retina growth & development, Xenopus Proteins metabolism, Zinc Finger Protein Gli3 metabolism
- Abstract
The Zic family of zinc finger transcription factors plays a critical role in multiple developmental processes. Using loss-of-function studies, we find that Zic5 is important for the differentiation of retinal pigmented epithelium (RPE) and the rod photoreceptor layer through suppressing Hedgehog (Hh) signaling. Further, Zic5 interacts with the critical Hh signaling molecule, Gli3, through the zinc finger domains of both proteins. This Zic5-Gli3 interaction disrupts Gli3/Gli3 homodimerization, resulting in Gli3 protein stabilization via a reduction in Gli3 ubiquitination. During embryonic Hh signaling, the activator form of Gli is normally converted to a repressor form through proteosome-mediated processing of Gli3, and the ratio of Gli3 repressor to full-length (activator) form of Gli3 determines the Gli3 repressor output required for normal eye development. Our results suggest Zic5 is a critical player in regulating Gli3 stability for the proper differentiation of RPE and rod photoreceptor layer during Xenopus eye development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)
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- 2022
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6. CEP97 phosphorylation by Dyrk1a is critical for centriole separation during multiciliogenesis.
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Lee M, Nagashima K, Yoon J, Sun J, Wang Z, Carpenter C, Lee HK, Hwang YS, Westlake CJ, and Daar IO
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- Animals, Cell Cycle Proteins metabolism, Cell Movement, Cytoskeletal Proteins chemistry, Embryo, Nonmammalian metabolism, Embryo, Nonmammalian ultrastructure, Humans, Phosphorylation, Protein Binding, Protein Serine-Threonine Kinases chemistry, Protein-Tyrosine Kinases chemistry, Proto-Oncogene Proteins metabolism, Substrate Specificity, Xenopus, Xenopus Proteins chemistry, Polo-Like Kinase 1, Centrioles metabolism, Cilia metabolism, Cytoskeletal Proteins metabolism, Organogenesis, Protein Serine-Threonine Kinases metabolism, Protein-Tyrosine Kinases metabolism, Xenopus Proteins metabolism
- Abstract
Proper cilia formation in multiciliated cells (MCCs) is necessary for appropriate embryonic development and homeostasis. Multicilia share many structural characteristics with monocilia and primary cilia, but there are still significant gaps in our understanding of the regulation of multiciliogenesis. Using the Xenopus embryo, we show that CEP97, which is known as a negative regulator of primary cilia formation, interacts with dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) to modulate multiciliogenesis. We show that Dyrk1a phosphorylates CEP97, which in turn promotes the recruitment of Polo-like kinase 1 (Plk1), which is a critical regulator of MCC maturation that functions to enhance centriole disengagement in cooperation with the enzyme Separase. Knockdown of either CEP97 or Dyrk1a disrupts cilia formation and centriole disengagement in MCCs, but this defect is rescued by overexpression of Separase. Thus, our study reveals that Dyrk1a and CEP97 coordinate with Plk1 to promote Separase function to properly form multicilia in vertebrate MCCs., (© 2021 Lee et al.)
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- 2022
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7. Characterization of a Compound Heterozygous SLC2A9 Mutation That Causes Hypouricemia.
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Yoon J, Cachau R, David VA, Thompson M, Jung W, Jee SH, Daar IO, Winkler CA, and Cho SK
- Abstract
Renal hypouricemia is a rare genetic disorder. Hypouricemia can present as renal stones or exercise-induced acute renal failure, but most cases are asymptomatic. Our previous study showed that two recessive variants of SLC22A12 (p.Trp258*, pArg90His) were identified in 90% of the hypouricemia patients from two independent cohorts: the Korean genome and epidemiology study (KoGES) and the Korean Cancer Prevention Study (KCPS-II). In this work, we investigate the genetic causes of hypouricemia in the rest of the 10% of unsolved cases. We found a novel non-synonymous mutation of SLC2A9 (voltage-sensitive uric acid transporter) in the whole-exome sequencing (WES) results. Molecular dynamics prediction suggests that the novel mutation p.Met126Val in SLCA9b (p.Met155Val in SLC2A9a) hinders uric acid transport through a defect of the outward open geometry. Molecular analysis using Xenopus oocytes confirmed that the p.Met126Val mutation significantly reduced uric acid transport but does not affect the SLC2A9 protein expression level. Our results will shed light on a better understanding of SLC2A9 -mediated uric acid transport and the development of a uric acid-lowering agent.
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- 2021
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8. Rab11fip5 regulates telencephalon development via ephrinB1 recycling.
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Yoon J, Garo J, Lee M, Sun J, Hwang YS, and Daar IO
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- Adaptor Proteins, Signal Transducing genetics, Animals, Cell Proliferation, Cytoskeleton, Endosomes metabolism, Ephrin-B1 genetics, Exocytosis, Gene Expression Regulation, Developmental, Gene Knockout Techniques, Neurogenesis, Telencephalon cytology, Xenopus laevis, Adaptor Proteins, Signal Transducing metabolism, Ephrin-B1 metabolism, Telencephalon growth & development, Telencephalon metabolism
- Abstract
Rab11 family-interacting protein 5 (Rab11fip5) is an adaptor protein that binds to the small GTPase Rab11, which has an important function in endosome recycling and trafficking of cellular proteins to the plasma membrane. Rab11fip5 is involved in many cellular processes, such as cytoskeleton rearrangement, iron uptake and exocytosis in neuroendocrine cells, and is also known as a candidate gene for autism-spectrum disorder. However, the role of Rab11fip5 during early embryonic development is not clearly understood. In this study, we identified Rab11fip5 as a protein that interacts with ephrinB1, a transmembrane ligand for Eph receptors. The PDZ binding motif in ephrinB1 and the Rab-binding domain in Rab11fip5 are necessary for their interaction in a complex. EphrinB1 and Rab11fip5 display overlapping expression in the telencephalon of developing amphibian embryos. The loss of Rab11fip5 function causes a reduction in telencephalon size and a decrease in the expression level of ephrinB1. Moreover, morpholino oligonucleotide-mediated knockdown of Rab11fip5 decreases cell proliferation in the telencephalon. The overexpression of ephrinB1 rescues these defects, suggesting that ephrinB1 recycling by the Rab11/Rab11fip5 complex is crucial for proper telencephalon development., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)
- Published
- 2021
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9. Sprouty2 regulates positioning of retinal progenitors through suppressing the Ras/Raf/MAPK pathway.
- Author
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Sun J, Yoon J, Lee M, Hwang YS, and Daar IO
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- Animals, Cell Differentiation physiology, Intracellular Signaling Peptides and Proteins metabolism, Neurulation physiology, Proto-Oncogene Proteins c-raf metabolism, Retina cytology, Xenopus Proteins metabolism, Intracellular Signaling Peptides and Proteins genetics, MAP Kinase Signaling System physiology, Retina embryology, Stem Cells cytology, Xenopus Proteins genetics, Xenopus laevis embryology
- Abstract
Sproutys are negative regulators of the Ras/Raf/MAPK signaling pathway and involved in regulation of organogenesis, differentiation, cell migration and proliferation. Although the function of Sproutys have been extensively studied during embryonic development, their role and mode of action during eye formation in vertebrate embryonic development is still unknown. Here we show that Xenopus sprouty2 is expressed in the optic vesicle at late neurula stage and knockdown of Sprouty2 prevents retinal progenitors from populating the retina, which in turn gives rise to small eyes. In the absence of Sprouty2, progenitor cell population of the retina can be restored by blocking the MAPK signaling pathway through overexpression of DN-Ras or DN-Raf. In contrast, activation of the MAPK pathway through overexpression of a constitutively active form of c-Raf (ca-Raf) inhibits progenitor population of the retina, similar to the Sprouty2 loss-of-function phenotype. Moreover, we present evidence that the retinal defect observed in Sprouty2 morphants is attributed to the failure of proper movement of retinal progenitors into the optic vesicle, rather than an effect on progenitor cell survival. These results suggest that Sprouty2 is required for the positioning of retinal progenitors within the optic vesicle through suppressing Ras/Raf/MAPK signaling pathway.
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- 2020
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10. Developmentally regulated GTP-binding protein 1 modulates ciliogenesis via an interaction with Dishevelled.
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Lee M, Hwang YS, Yoon J, Sun J, Harned A, Nagashima K, and Daar IO
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- Actins metabolism, Adaptor Proteins, Signal Transducing metabolism, Animals, Basal Bodies metabolism, Cell Line, Cell Polarity, Dishevelled Proteins chemistry, Embryo, Nonmammalian metabolism, GTP-Binding Proteins chemistry, Humans, Phenotype, Protein Binding, Protein Domains, Protein Transport, Xenopus Proteins chemistry, Xenopus laevis embryology, Cilia metabolism, Dishevelled Proteins metabolism, GTP-Binding Proteins metabolism, Organogenesis, Xenopus Proteins metabolism, Xenopus laevis metabolism
- Abstract
Cilia are critical for proper embryonic development and maintaining homeostasis. Although extensively studied, there are still significant gaps regarding the proteins involved in regulating ciliogenesis. Using the Xenopus laevis embryo, we show that Dishevelled (Dvl), a key Wnt signaling scaffold that is critical to proper ciliogenesis, interacts with Drg1 (developmentally regulated GTP-binding protein 1). The loss of Drg1 or disruption of the interaction with Dvl reduces the length and number of cilia and displays defects in basal body migration and docking to the apical surface of multiciliated cells (MCCs). Moreover, Drg1 morphants display abnormal rotational polarity of basal bodies and a decrease in apical actin and RhoA activity that can be attributed to disruption of the protein complex between Dvl and Daam1, as well as between Daam1 and RhoA. These results support the concept that the Drg1-Dvl interaction regulates apical actin polymerization and stability in MCCs. Thus, Drg1 is a newly identified partner of Dvl in regulating ciliogenesis., (© 2019 Lee et al.)
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- 2019
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11. TBC1d24-ephrinB2 interaction regulates contact inhibition of locomotion in neural crest cell migration.
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Yoon J, Hwang YS, Lee M, Sun J, Cho HJ, Knapik L, and Daar IO
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- Animals, Blotting, Western, Carrier Proteins genetics, Cell Movement genetics, Cell Movement physiology, Ephrin-B2 genetics, GTPase-Activating Proteins, Immunoprecipitation, Locomotion genetics, Locomotion physiology, Membrane Proteins, Microscopy, Fluorescence, Nerve Tissue Proteins, Phosphorylation, Protein Binding, Xenopus, Carrier Proteins metabolism, Embryo, Nonmammalian cytology, Embryo, Nonmammalian metabolism, Ephrin-B2 metabolism, Neural Crest cytology, Neural Crest metabolism
- Abstract
Although Eph-ephrin signalling has been implicated in the migration of cranial neural crest (CNC) cells, it is still unclear how ephrinB transduces signals regulating this event. We provide evidence that TBC1d24, a putative Rab35-GTPase activating protein (Rab35 GAP), complexes with ephrinB2 via the scaffold Dishevelled (Dsh) and mediates a signal affecting contact inhibition of locomotion (CIL) in CNC cells. Moreover, we found that, in migrating CNC, the interaction between ephrinB2 and TBC1d24 negatively regulates E-cadherin recycling in these cells via Rab35. Upon engagement of the cognate Eph receptor, ephrinB2 is tyrosine phosphorylated, which disrupts the ephrinB2/Dsh/TBC1d24 complex. The dissolution of this complex leads to increasing E-cadherin levels at the plasma membrane, resulting in loss of CIL and disrupted CNC migration. Our results indicate that TBC1d24 is a critical player in ephrinB2 control of CNC cell migration via CIL.
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- 2018
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12. EphrinB1 promotes cancer cell migration and invasion through the interaction with RhoGDI1.
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Cho HJ, Hwang YS, Yoon J, Lee M, Lee HG, and Daar IO
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- Apoptosis, Biomarkers, Tumor genetics, Cell Proliferation, Humans, Neoplasm Invasiveness, Neoplasms genetics, Neoplasms metabolism, Receptor, EphB2 genetics, Tumor Cells, Cultured, rho Guanine Nucleotide Dissociation Inhibitor alpha genetics, rhoA GTP-Binding Protein genetics, Biomarkers, Tumor metabolism, Cell Movement, Neoplasms pathology, Receptor, EphB2 metabolism, rho Guanine Nucleotide Dissociation Inhibitor alpha metabolism, rhoA GTP-Binding Protein metabolism
- Abstract
Eph receptors and their corresponding ephrin ligands have been associated with regulating cell-cell adhesion and motility, and thus have a critical role in various biological processes including tissue morphogenesis and homeostasis, as well as pathogenesis of several diseases. Aberrant regulation of Eph/ephrin signaling pathways is implicated in tumor progression of various human cancers. Here, we show that a Rho family GTPase regulator, Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1), can interact with ephrinB1, and this interaction is enhanced upon binding the extracellular domain of the cognate EphB2 receptor. Deletion mutagenesis revealed that amino acids 327-334 of the ephrinB1 intracellular domain are critical for the interaction with RhoGDI1. Stimulation with an EphB2 extracellular domain-Fc fusion protein (EphB2-Fc) induces RhoA activation and enhances the motility as well as invasiveness of wild-type ephrinB1-expressing cells. These Eph-Fc-induced effects were markedly diminished in cells expressing the mutant ephrinB1 construct (Δ327-334) that is ineffective at interacting with RhoGDI1. Furthermore, ephrinB1 depletion by siRNA suppresses EphB2-Fc-induced RhoA activation, and reduces motility and invasiveness of the SW480 and Hs578T human cancer cell lines. Our study connects the interaction between RhoGDI1 and ephrinB1 to the promotion of cancer cell behavior associated with tumor progression. This interaction may represent a therapeutic target in cancers that express ephrinB1.
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- 2018
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13. A frog's view of EphrinB signaling.
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Hwang YS and Daar IO
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- Animals, Ephrins biosynthesis, Gene Expression Regulation, Developmental, Humans, Models, Animal, Receptors, Eph Family biosynthesis, Signal Transduction genetics, Xenopus genetics, Xenopus growth & development, Cell Adhesion genetics, Embryonic Development genetics, Ephrins genetics, Receptors, Eph Family genetics
- Abstract
Cell-cell and cell-substrate adhesion are essential to the proper formation and maintenance of tissue patterns during development, and deregulation of these processes can lead to invasion and metastasis of cancer cells. Cell surface adhesion and signaling molecules are key players in both normal development and cancer progression. One set of cell surface proteins, the Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, are significant regulators of these processes. During embryonic development, the Eph/ephrin signaling system is involved in cell-cell contact events that result in cell sorting and boundary formation between receptor and ligand bearing cells. When migrating cells that display the membrane bound ligands or receptors come in contact with cells bearing the cognate partner, the response may be adhesion or repulsion, ultimately leading to the proper positioning of these cells. During cancer progression, the signaling between these receptor/ligand pairs is often deregulated, leading to increased invasion and metastasis. To gain mechanistic insight into the pathways that mediate Eph receptor and ephrin signaling we have relied upon a very tractable system, the frog Xenopus. This model system has proven to be extremely versatile, and represents a relatively quick and manipulable system to explore signaling events and the in vivo processes affected by these signals., (© 2017 Wiley Periodicals, Inc.)
- Published
- 2017
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14. Neural transcription factors bias cleavage stage blastomeres to give rise to neural ectoderm.
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Gaur S, Mandelbaum M, Herold M, Majumdar HD, Neilson KM, Maynard TM, Mood K, Daar IO, and Moody SA
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- Animals, Blastomeres metabolism, Blastula growth & development, Ectoderm metabolism, Forkhead Transcription Factors biosynthesis, Gastrula growth & development, Gene Expression Regulation, Developmental, Humans, Mice, Neural Plate metabolism, Transcription Factors biosynthesis, Transcription Factors genetics, Xenopus Proteins biosynthesis, Xenopus Proteins genetics, Xenopus laevis genetics, Xenopus laevis growth & development, Zygote growth & development, Cell Differentiation genetics, Ectoderm growth & development, Forkhead Transcription Factors genetics, Neural Plate growth & development
- Abstract
The decision by embryonic ectoderm to give rise to epidermal versus neural derivatives is the result of signaling events during blastula and gastrula stages. However, there also is evidence in Xenopus that cleavage stage blastomeres contain maternally derived molecules that bias them toward a neural fate. We used a blastomere explant culture assay to test whether maternally deposited transcription factors bias 16-cell blastomere precursors of epidermal or neural ectoderm to express early zygotic neural genes in the absence of gastrulation interactions or exogenously supplied signaling factors. We found that Foxd4l1, Zic2, Gmnn, and Sox11 each induced explants made from ventral, epidermis-producing blastomeres to express early neural genes, and that at least some of the Foxd4l1 and Zic2 activities are required at cleavage stages. Similarly, providing extra Foxd4l1 or Zic2 to explants made from dorsal, neural plate-producing blastomeres significantly increased the expression of early neural genes, whereas knocking down either significantly reduced them. These results show that maternally delivered transcription factors bias cleavage stage blastomeres to a neural fate. We demonstrate that mouse and human homologs of Foxd4l1 have similar functional domains compared to the frog protein, as well as conserved transcriptional activities when expressed in Xenopus embryos and blastomere explants. genesis 54:334-349, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)
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- 2016
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15. Early steps in primary cilium assembly require EHD1/EHD3-dependent ciliary vesicle formation.
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Lu Q, Insinna C, Ott C, Stauffer J, Pintado PA, Rahajeng J, Baxa U, Walia V, Cuenca A, Hwang YS, Daar IO, Lopes S, Lippincott-Schwartz J, Jackson PK, Caplan S, and Westlake CJ
- Published
- 2015
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16. EphrinB1 interacts with CNK1 and promotes cell migration through c-Jun N-terminal kinase (JNK) activation.
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Cho HJ, Hwang YS, Mood K, Ji YJ, Lim J, Morrison DK, and Daar IO
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- Cell Line, Cell Line, Tumor, Enzyme Activation, Ephrin-B1 genetics, Humans, Intracellular Signaling Peptides and Proteins genetics, JNK Mitogen-Activated Protein Kinases genetics, Phosphorylation, Protein Binding, rhoA GTP-Binding Protein genetics, rhoA GTP-Binding Protein metabolism, Cell Movement, Ephrin-B1 metabolism, Intracellular Signaling Peptides and Proteins metabolism, JNK Mitogen-Activated Protein Kinases metabolism
- Abstract
The Eph receptors and their membrane-bound ligands, ephrins, play important roles in various biological processes such as cell adhesion and movement. The transmembrane ephrinBs transduce reverse signaling in a tyrosine phosphorylation-dependent or -independent, as well as PDZ-dependent manner. Here, we show that ephrinB1 interacts with Connector Enhancer of KSR1 (CNK1) in an EphB receptor-independent manner. In cultured cells, cotransfection of ephrinB1 with CNK1 increases JNK phosphorylation. EphrinB1/CNK1-mediated JNK activation is reduced by overexpression of dominant-negative RhoA. Overexpression of CNK1 alone is sufficient for activation of RhoA; however, both ephrinB1 and CNK1 are required for JNK phosphorylation. Co-immunoprecipitation data showed that ephrinB1 and CNK1 act as scaffold proteins that connect RhoA and JNK signaling components, such as p115RhoGEF and MKK4. Furthermore, adhesion to fibronectin or active Src overexpression increases ephrinB1/CNK1 binding, whereas blocking Src activity by a pharmacological inhibitor decreases not only ephrinB1/CNK1 binding, but also JNK activation. EphrinB1 overexpression increases cell motility, however, CNK1 depletion by siRNA abrogates ephrinB1-mediated cell migration and JNK activation. Moreover, Rho kinase inhibitor or JNK inhibitor treatment suppresses ephrinB1-mediated cell migration. Taken together, our findings suggest that CNK1 is required for ephrinB1-induced JNK activation and cell migration., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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17. EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1.
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Ji YJ, Hwang YS, Mood K, Cho HJ, Lee HS, Winterbottom E, Cousin H, and Daar IO
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- Animals, Blotting, Western, Embryo, Nonmammalian physiology, Immunoprecipitation, Microscopy, Fluorescence, Neural Tube Defects metabolism, Xenopus laevis metabolism, ADAM Proteins metabolism, Ephrin-B2 metabolism, Membrane Proteins metabolism, Morphogenesis physiology, Neural Tube Defects embryology, Signal Transduction physiology, Xenopus laevis embryology
- Abstract
The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.
- Published
- 2014
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18. Abelson interactor 1 (ABI1) and its interaction with Wiskott-Aldrich syndrome protein (wasp) are critical for proper eye formation in Xenopus embryos.
- Author
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Singh A, Winterbottom EF, Ji YJ, Hwang YS, and Daar IO
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- Actin Cytoskeleton metabolism, Adaptor Proteins, Signal Transducing chemistry, Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing physiology, Animals, Cell Lineage, Cell Movement, Open Reading Frames, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Retina embryology, Signal Transduction, Stem Cells cytology, Xenopus genetics, Xenopus Proteins chemistry, src Homology Domains, Adaptor Proteins, Signal Transducing metabolism, Eye embryology, Gene Expression Regulation, Developmental, Wiskott-Aldrich Syndrome Protein metabolism, Xenopus embryology, Xenopus Proteins physiology
- Abstract
Abl interactor 1 (Abi1) is a scaffold protein that plays a central role in the regulation of actin cytoskeleton dynamics as a constituent of several key protein complexes, and homozygous loss of this protein leads to embryonic lethality in mice. Because this scaffold protein has been shown in cultured cells to be a critical component of pathways controlling cell migration and actin regulation at cell-cell contacts, we were interested to investigate the in vivo role of Abi1 in morphogenesis during the development of Xenopus embryos. Using morpholino-mediated translation inhibition, we demonstrate that knockdown of Abi1 in the whole embryo, or specifically in eye field progenitor cells, leads to disruption of eye morphogenesis. Moreover, signaling through the Src homology 3 domain of Abi1 is critical for proper movement of retinal progenitor cells into the eye field and their appropriate differentiation, and this process is dependent upon an interaction with the nucleation-promoting factor Wasp (Wiskott-Aldrich syndrome protein). Collectively, our data demonstrate that the Abi1 scaffold protein is an essential regulator of cell movement processes required for normal eye development in Xenopus embryos and specifically requires an Src homology 3 domain-dependent interaction with Wasp to regulate this complex morphogenetic process.
- Published
- 2013
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19. Conserved structural domains in FoxD4L1, a neural forkhead box transcription factor, are required to repress or activate target genes.
- Author
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Klein SL, Neilson KM, Orban J, Yaklichkin S, Hoffbauer J, Mood K, Daar IO, and Moody SA
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- Amino Acid Motifs, Amino Acid Sequence, Animals, Hydrogen-Ion Concentration, Mice, Molecular Sequence Data, Protein Structure, Tertiary, Xenopus Proteins chemistry, Xenopus Proteins metabolism, Computational Biology, Conserved Sequence, Forkhead Transcription Factors chemistry, Forkhead Transcription Factors metabolism, Gene Silencing, Transcriptional Activation
- Abstract
FoxD4L1 is a forkhead transcription factor that expands the neural ectoderm by down-regulating genes that promote the onset of neural differentiation and up-regulating genes that maintain proliferative neural precursors in an immature state. We previously demonstrated that binding of Grg4 to an Eh-1 motif enhances the ability of FoxD4L1 to down-regulate target neural genes but does not account for all of its repressive activity. Herein we analyzed the protein sequence for additional interaction motifs and secondary structure. Eight conserved motifs were identified in the C-terminal region of fish and frog proteins. Extending the analysis to mammals identified a high scoring motif downstream of the Eh-1 domain that contains a tryptophan residue implicated in protein-protein interactions. In addition, secondary structure prediction programs predicted an α-helical structure overlapping with amphibian-specific Motif 6 in Xenopus, and similarly located α-helical structures in other vertebrate FoxD proteins. We tested functionality of this site by inducing a glutamine-to-proline substitution expected to break the predicted α-helical structure; this significantly reduced FoxD4L1's ability to repress zic3 and irx1. Because this mutation does not interfere with Grg4 binding, these results demonstrate that at least two regions, the Eh-1 motif and a more C-terminal predicted α-helical/Motif 6 site, additively contribute to repression. In the N-terminal region we previously identified a 14 amino acid motif that is required for the up-regulation of target genes. Secondary structure prediction programs predicted a short β-strand separating two acidic domains. Mutant constructs show that the β-strand itself is not required for transcriptional activation. Instead, activation depends upon a glycine residue that is predicted to provide sufficient flexibility to bring the two acidic domains into close proximity. These results identify conserved predicted motifs with secondary structures that enable FoxD4L1 to carry out its essential functions as both a transcriptional repressor and activator of neural genes.
- Published
- 2013
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20. The Smurf ubiquitin ligases regulate tissue separation via antagonistic interactions with ephrinB1.
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Hwang YS, Lee HS, Kamata T, Mood K, Cho HJ, Winterbottom E, Ji YJ, Singh A, and Daar IO
- Subjects
- Animals, Embryo, Nonmammalian enzymology, Gene Expression Regulation, Developmental, Gene Knockdown Techniques, Monomeric GTP-Binding Proteins metabolism, Ubiquitin-Protein Ligases genetics, Zebrafish Proteins metabolism, Ephrin-B1 metabolism, Ubiquitin-Protein Ligases metabolism, Xenopus genetics, Xenopus metabolism
- Abstract
The formation of tissue boundaries is dependent on the cell-cell adhesion/repulsion system that is required for normal morphogenetic processes during development. The Smad ubiquitin regulatory factors (Smurfs) are E3 ubiquitin ligases with established roles in cell growth and differentiation, but whose roles in regulating cell adhesion and migration are just beginning to emerge. Here, we demonstrate that the Smurfs regulate tissue separation at mesoderm/ectoderm boundaries through antagonistic interactions with ephrinB1, an Eph receptor ligand that has a key role in regulating the separation of embryonic germ layers. EphrinB1 is targeted by Smurf2 for degradation; however, a Smurf1 interaction with ephrinB1 prevents the association with Smurf2 and precludes ephrinB1 from ubiquitination and degradation, since it is a substantially weaker substrate for Smurf1. Inhibition of Smurf1 expression in embryonic mesoderm results in loss of ephrinB1-mediated separation of this tissue from the ectoderm, which can be rescued by the coincident inhibition of Smurf2 expression. This system of differential interactions between Smurfs and ephrinB1 regulates the maintenance of tissue boundaries through the control of ephrinB protein levels.
- Published
- 2013
- Full Text
- View/download PDF
21. Specific domains of FoxD4/5 activate and repress neural transcription factor genes to control the progression of immature neural ectoderm to differentiating neural plate.
- Author
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Neilson KM, Klein SL, Mhaske P, Mood K, Daar IO, and Moody SA
- Subjects
- Amino Acid Sequence, Animals, Cell Differentiation, Ectoderm cytology, Ectoderm metabolism, Forkhead Transcription Factors genetics, Forkhead Transcription Factors metabolism, Humans, Mice, Molecular Sequence Data, Neural Plate cytology, Neural Plate metabolism, Protein Structure, Tertiary, Xenopus, Xenopus Proteins genetics, Xenopus Proteins metabolism, Ectoderm embryology, Forkhead Transcription Factors chemistry, Gene Expression Regulation, Developmental, Neural Plate embryology, Transcriptional Activation, Xenopus Proteins chemistry
- Abstract
FoxD4/5, a forkhead transcription factor, plays a critical role in establishing and maintaining the embryonic neural ectoderm. It both up-regulates genes that maintain a proliferative, immature neural ectoderm and down-regulates genes that promote the transition to a differentiating neural plate. We constructed deletion and mutant versions of FoxD4/5 to determine which domains are functionally responsible for these opposite activities, which regulate the critical developmental transition of neural precursors to neural progenitors to differentiating neural plate cells. Our results show that up-regulation of genes that maintain immature neural precursors (gem, zic2) requires the Acidic blob (AB) region in the N-terminal portion of the protein, indicating that the AB is the transactivating domain. Additionally, down-regulation of those genes that promote the transition to neural progenitors (sox) and those that lead to neural differentiation (zic, irx) involves: 1) an interaction with the Groucho co-repressor at the Eh-1 motif in the C-terminus; and 2) sequence downstream of this motif. Finally, the ability of FoxD4/5 to induce the ectopic expression of neural precursor genes in the ventral ectoderm also involves both the AB region and the Eh-1 motif; FoxD4/5 accomplishes ectopic neural induction by both activating neural precursor genes and repressing BMP signaling and epidermal genes. This study identifies the specific, conserved domains of the FoxD4/5 protein that allow this single transcription factor to regulate a network of genes that controls the transition of a proliferative neural ectodermal population to a committed neural plate population poised to begin differentiation., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
22. Non-SH2/PDZ reverse signaling by ephrins.
- Author
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Daar IO
- Subjects
- Animals, Cell Adhesion, Cell Movement, Ephrins metabolism, Humans, Neoplasms metabolism, Neoplasms pathology, PDZ Domains, Receptors, Eph Family metabolism, src Homology Domains, Ephrins physiology, Signal Transduction
- Abstract
Great strides have been made regarding our understanding of the processes and signaling events influenced by Eph/ephrin signaling that play a role in cell adhesion and cell movement. However, the precise mechanisms by which these signaling events regulate cell and tissue architecture still need further resolution. The Eph/ephrin signaling pathways and the ability to regulate cell-cell adhesion and motility constitutes an impressive system for regulating tissue separation and morphogenesis (Pasquale, 2005, 2008 [1,2]). Moreover, the de-regulation of this signaling system is linked to the promotion of aggressive and metastatic tumors in humans [2]. In the following section, we discuss some of the interesting mechanisms by which ephrins can signal through their own intracellular domains (reverse signaling) either independent of forward signaling or in addition to forward signaling through a cognate receptor. In this review we discuss how ephrins (Eph ligands) "reverse signal" through their intracellular domains to affect cell adhesion and movement, but the focus is on modes of action that are independent of SH2 and PDZ interactions., (Published by Elsevier Ltd.)
- Published
- 2012
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- View/download PDF
23. Using 32-cell stage Xenopus embryos to probe PCP signaling.
- Author
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Lee HS, Sokol SY, Moody SA, and Daar IO
- Subjects
- Animals, Blastomeres cytology, Female, Fluorescent Antibody Technique, Male, Tissue Embedding, Tissue Fixation, beta-Galactosidase metabolism, Cell Polarity, Embryo, Nonmammalian cytology, Signal Transduction, Xenopus embryology
- Abstract
Use of loss-of function (via antisense Morpholino oligonucleotides (MOs)) or over-expression of proteins in epithelial cells during early embryogenesis of Xenopus embryos, can be a powerful tool to understand how signaling molecules can affect developmental events. The techniques described here are useful for examining the roles of proteins in cell-cell adhesion, and planar cell polarity (PCP) signaling in cell movement. We describe how to target specific regions within the embryos by injecting an RNA encoding a tracer molecule along with RNA encoding your protein of interest or an antisense MO to knock-down a particular protein within a specific blastomere of the embryo. Effects on cell-cell adhesion, cell movement, and endogenous or exogenous protein localization can be assessed at later stages in specific targeted tissues using fluorescent microscopy and immunolocalization.
- Published
- 2012
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- View/download PDF
24. Human cancer. Preface.
- Author
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Daar IO
- Subjects
- Disease Progression, Humans, Tumor Microenvironment, Cell Transformation, Neoplastic metabolism, Cell Transformation, Neoplastic pathology, Neoplasms metabolism, Neoplasms pathology
- Published
- 2012
- Full Text
- View/download PDF
25. Eph/ephrin signaling in cell-cell and cell-substrate adhesion.
- Author
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Singh A, Winterbottom E, and Daar IO
- Subjects
- Animals, Dendrites physiology, Extracellular Matrix physiology, Humans, Models, Biological, Neuronal Plasticity physiology, Signal Transduction, Cell Adhesion physiology, Ephrins physiology, Receptors, Eph Family physiology
- Abstract
Cell-cell and cell-matrix adhesion are critical processes for the formation and maintenance of tissue patterns during development, as well as control of invasion and metastasis of cancer cells. Although great strides have been made regarding our understanding of the processes that play a role in cell adhesion and cell movement, the precise mechanisms by which diverse signaling events regulate cell and tissue architecture are poorly understood. One group of cell surface molecules, Eph receptor tyrosine kinases, and their membrane-bound ligands, ephrins, are key regulators in these processes. It is the ability of Eph/ephrin signaling pathways to regulate cell-cell adhesion and motility that establishes this family as a formidable system for regulating tissue separation and morphogenesis. Moreover, the de-regulation of this signaling system is linked to the promotion of more aggressive and metastatic tumors in humans.
- Published
- 2012
- Full Text
- View/download PDF
26. Functional coupling between the extracellular matrix and nuclear lamina by Wnt signaling in progeria.
- Author
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Hernandez L, Roux KJ, Wong ES, Mounkes LC, Mutalif R, Navasankari R, Rai B, Cool S, Jeong JW, Wang H, Lee HS, Kozlov S, Grunert M, Keeble T, Jones CM, Meta MD, Young SG, Daar IO, Burke B, Perantoni AO, and Stewart CL
- Subjects
- Animals, Apoptosis, Biomarkers metabolism, Blotting, Western, Cell Proliferation, Cells, Cultured, Chromatin Immunoprecipitation, Embryo, Mammalian cytology, Embryo, Mammalian metabolism, Enzyme-Linked Immunosorbent Assay, Fibroblasts cytology, Fibroblasts metabolism, Gene Expression Profiling, Humans, Lamin Type A physiology, Luciferases metabolism, Mice, Mice, Knockout, Oligonucleotide Array Sequence Analysis, Progeria pathology, RNA, Messenger genetics, Reverse Transcriptase Polymerase Chain Reaction, Syndrome, Extracellular Matrix metabolism, Nuclear Lamina metabolism, Progeria metabolism, Signal Transduction physiology, Wnt Proteins metabolism, Xenopus laevis embryology
- Abstract
The segmental premature aging disease Hutchinson-Gilford Progeria (HGPS) is caused by a truncated and farnesylated form of Lamin A. In a mouse model for HGPS, a similar Lamin A variant causes the proliferative arrest and death of postnatal, but not embryonic, fibroblasts. Arrest is due to an inability to produce a functional extracellular matrix (ECM), because growth on normal ECM rescues proliferation. The defects are associated with inhibition of canonical Wnt signaling, due to reduced nuclear localization and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. Defective Wnt signaling, affecting ECM synthesis, may be critical to the etiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. These results establish a functional link between the nuclear envelope/lamina and the cell surface/ECM and may provide insights into the role of Wnt signaling and the ECM in aging., (Copyright © 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
27. EphrinB reverse signaling in cell-cell adhesion: is it just par for the course?
- Author
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Lee HS and Daar IO
- Subjects
- Animals, Cell Adhesion, Humans, Models, Biological, Tight Junctions metabolism, Adaptor Proteins, Signal Transducing metabolism, Ephrin-B1 metabolism, Signal Transduction
- Abstract
Cell-cell adhesion is a critical process for the formation and maintenance of tissue patterns during development, as well as invasion and metastasis of cancer cells. Although great strides have been made regarding our understanding of the processes that play a role in cell-cell adhesion, the precise mechanisms by which diverse signaling events regulate cell and tissue architecture is poorly understood. In this commentary we will focus on the Eph/ephrin signaling system, and specifically how the ephrinB1 transmembrane ligand for Eph receptor tyrosine kinases sends signals affecting cell-cell junctions. In a recent study using the epithelial cells of early stage Xenopus embryos, we have shown that loss- or gain-of function of ephrinB1 can disrupt cell-cell contacts and tight junctions. This study reveals a mechanism where ephrinB1 competes with active Cdc42 for binding to Par-6, a scaffold protein central to the Par polarity complex (Par-3/Par-6/Cdc42/aPKC) and disrupts the localization of tight junction-associated proteins (ZO-1, Cingulin) at tight junctions. This competition reduces aPKC activity critical to maintaining and/or forming tight junctions. Finally, phosphorylation of ephrinB1 on specific tyrosine residues can block the interaction between ephrinB1 and Par-6 at tight junctions, and restore tight junction formation. Recent evidence indicates that de-regulation of forward signaling through EphB receptors may play a role in metastatic progression in colon cancer. In light of the new data showing an effect of ephrinB reverse signaling on tight junctions, an additional mechanism can be hypothesized where de-regulation of ephrinB1 expression or phosphorylation may also impact metastatic progression.
- Published
- 2009
- Full Text
- View/download PDF
28. Fibroblast growth factor receptor-induced phosphorylation of ephrinB1 modulates its interaction with Dishevelled.
- Author
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Lee HS, Mood K, Battu G, Ji YJ, Singh A, and Daar IO
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Amino Acid Sequence, Animals, Dishevelled Proteins, Ephrin-B1 genetics, Humans, Molecular Sequence Data, Phosphoproteins genetics, Phosphorylation, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Retina cytology, Retina embryology, Signal Transduction physiology, Stem Cells cytology, Stem Cells metabolism, Tyrosine metabolism, Xenopus Proteins genetics, Xenopus laevis anatomy & histology, Adaptor Proteins, Signal Transducing metabolism, Ephrin-B1 metabolism, Phosphoproteins metabolism, Receptors, Fibroblast Growth Factor metabolism, Xenopus Proteins metabolism, Xenopus laevis embryology, Xenopus laevis metabolism
- Abstract
The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB1 protein is a bidirectional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB1 plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). However, the mechanism by which the FGFR may regulate this cell movement is unknown. Here, we present evidence that FGFR-induced repression of retinal fate is dependent upon phosphorylation within the intracellular domain of ephrinB1. We demonstrate that phosphorylation of tyrosines 324 and 325 disrupts the ephrinB1/Dsh interaction, thus modulating retinal progenitor movement that is dependent on the planar cell polarity pathway. These results provide mechanistic insight into how fibroblast growth factor signaling modulates ephrinB1 control of retinal progenitor movement within the eye field.
- Published
- 2009
- Full Text
- View/download PDF
29. EphrinB1 controls cell-cell junctions through the Par polarity complex.
- Author
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Lee HS, Nishanian TG, Mood K, Bong YS, and Daar IO
- Subjects
- Animals, Embryo, Nonmammalian, Ephrin-B1 metabolism, Phosphorylation, Protein Binding, Tight Junctions, Xenopus laevis, Ephrin-B1 physiology, Intercellular Junctions, Xenopus Proteins physiology
- Abstract
A body of evidence is emerging that shows a requirement for ephrin ligands in the proper migration of cells, and the formation of cell and tissue boundaries. These processes are dependent on the cell-cell adhesion system, which plays a crucial role in normal morphogenetic processes during development, as well as in invasion and metastasis. Although ephrinB ligands are bi-directional signalling molecules, the precise mechanism by which ephrinB1 signals through its intracellular domain to regulate cell-cell adhesion in epithelial cells remains unclear. Here, we present evidence that ephrinB1 associates with the Par polarity complex protein Par-6 (a scaffold protein required for establishing tight junctions) and can compete with the small GTPase Cdc42 for association with Par-6. This competition causes inactivation of the Par complex, resulting in the loss of tight junctions. Moreover, the interaction between ephrinB1 and Par-6 is disrupted by tyrosine phosphorylation of the intracellular domain of ephrinB1. Thus, we have identified a mechanism by which ephrinB1 signalling regulates cell-cell junctions in epithelial cells, and this may influence how we devise therapeutic interventions regarding these molecules in metastatic disease.
- Published
- 2008
- Full Text
- View/download PDF
30. ephrinB1 signals from the cell surface to the nucleus by recruitment of STAT3.
- Author
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Bong YS, Lee HS, Carim-Todd L, Mood K, Nishanian TG, Tessarollo L, and Daar IO
- Subjects
- Active Transport, Cell Nucleus, Amino Acid Sequence, Animals, Cell Line, Chlorocebus aethiops, DNA metabolism, Ephrin-B1 chemistry, Ephrin-B1 genetics, Humans, Janus Kinase 2 genetics, Janus Kinase 2 metabolism, Molecular Sequence Data, Phosphorylation, Phosphotyrosine metabolism, Protein Binding, Rats, STAT3 Transcription Factor genetics, Transcription, Genetic genetics, Xenopus laevis, Cell Membrane metabolism, Cell Nucleus metabolism, Ephrin-B1 metabolism, STAT3 Transcription Factor metabolism, Signal Transduction
- Abstract
The Eph (erythropoietin-producing hepatoma) family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB (Eph receptor interactor B) protein is a bidirectional signaling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase, residing on another cell. A reverse signal can be transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Although some insight has been gained regarding how ephrinB may send signals affecting cytoskeletal components, little is known about how ephrinB1 reverse signaling affects transcriptional processes. Here we report that signal transducer and activator of transcription 3 (STAT3) can interact with ephrinB1 in a phosphorylation-dependent manner that leads to enhanced activation of STAT3 transcriptional activity. This activity depends on the tyrosine kinase Jak2, and two tyrosines within the intracellular domain of ephrinB1 are critical for the association with STAT3 and its activation. The recruitment of STAT3 to ephrinB1, and its resulting Jak2-dependent activation and transcription of reporter targets, reveals a signaling pathway from ephrinB1 to the nucleus.
- Published
- 2007
- Full Text
- View/download PDF
31. Gab1 is required for cell cycle transition, cell proliferation, and transformation induced by an oncogenic met receptor.
- Author
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Mood K, Saucier C, Bong YS, Lee HS, Park M, and Daar IO
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Binding Sites, Cell Proliferation, Cells, Cultured, Conserved Sequence, Enzyme Activation drug effects, Fibroblasts cytology, Fibroblasts drug effects, Humans, Intracellular Signaling Peptides and Proteins metabolism, JNK Mitogen-Activated Protein Kinases metabolism, Mitogen-Activated Protein Kinases metabolism, Molecular Sequence Data, Oncogene Protein tpr-met pharmacology, Oocytes cytology, Oocytes drug effects, Phosphatidylinositol 3-Kinases metabolism, Phosphoproteins chemistry, Progesterone pharmacology, Protein Structure, Tertiary, Protein Transport drug effects, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatases metabolism, Receptors, Fibroblast Growth Factor metabolism, Cell Cycle, Cell Transformation, Neoplastic, Phosphoproteins metabolism, Proto-Oncogene Proteins c-met metabolism
- Abstract
We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cgamma binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met-mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor.
- Published
- 2006
- Full Text
- View/download PDF
32. Oncogenic Met receptor induces ectopic structures in Xenopus embryos.
- Author
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Ishimura A, Lee HS, Bong YS, Saucier C, Mood K, Park EK, and Daar IO
- Subjects
- Animals, Female, Mutation, Phosphorylation, Proto-Oncogene Proteins c-met genetics, Xenopus, Embryo, Nonmammalian metabolism, Embryonic Induction physiology, Proto-Oncogene Proteins c-met physiology
- Abstract
When aberrantly expressed or activated, the Met receptor tyrosine kinase is involved in tumor invasiveness and metastasis. In this study, we have used the Xenopus embryonic system to define the role of various Met proximal-binding partners and downstream signaling pathways in regulating an induced morphogenetic event. We show that expression of an oncogenic derivative of the Met receptor (Tpr-Met) induces ectopic morphogenetic structures during Xenopus embryogenesis. Using variant forms of Tpr-Met that are engineered to recruit a specific signaling molecule of choice, we demonstrate that the sole recruitment of either the Grb2 or the Shc adaptor protein is sufficient to induce ectopic structures and anterior reduction, while the recruitment of PI-3Kinase (PI-3K) is necessary but not sufficient for this effect. In contrast, the recruitment of PLCgamma can initiate the induction, but fails to maintain or elongate supernumerary structures. Finally, evidence indicates that the Ras/Raf/MAPK pathway is necessary, but not sufficient to induce these structures. This study also emphasizes the importance of examining signaling molecules in the regulatory context that is provided by receptor/effector interactions when assessing a role in cell growth and differentiation.
- Published
- 2006
- Full Text
- View/download PDF
33. Oncogenic Met receptor induces cell-cycle progression in Xenopus oocytes independent of direct Grb2 and Shc binding or Mos synthesis, but requires phosphatidylinositol 3-kinase and Raf signaling.
- Author
-
Mood K, Saucier C, Ishimura A, Bong YS, Lee HS, Park M, and Daar IO
- Subjects
- Adaptor Proteins, Signal Transducing genetics, Adaptor Proteins, Signal Transducing metabolism, Animals, CDC2 Protein Kinase metabolism, Cell Cycle genetics, Chromones pharmacology, Enzyme Inhibitors pharmacology, Extracellular Signal-Regulated MAP Kinases metabolism, Female, GRB2 Adaptor Protein genetics, GRB2 Adaptor Protein metabolism, JNK Mitogen-Activated Protein Kinases metabolism, MAP Kinase Kinase Kinases antagonists & inhibitors, MAP Kinase Kinase Kinases metabolism, Mitogen-Activated Protein Kinase 8 genetics, Mitogen-Activated Protein Kinase 8 metabolism, Models, Biological, Morpholines pharmacology, Mutation, Oocytes drug effects, Oocytes metabolism, Phosphatidylinositol 3-Kinases genetics, Phosphoinositide-3 Kinase Inhibitors, Phospholipase C gamma genetics, Phospholipase C gamma metabolism, Phosphorylation, Progesterone pharmacology, Protein Binding, Proto-Oncogene Proteins c-met genetics, Proto-Oncogene Proteins c-met metabolism, Proto-Oncogene Proteins c-mos genetics, Proto-Oncogene Proteins c-mos metabolism, Proto-Oncogene Proteins c-raf genetics, RNA, Antisense genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Shc Signaling Adaptor Proteins, Signal Transduction, Src Homology 2 Domain-Containing, Transforming Protein 1, Transfection, Xenopus Proteins genetics, Xenopus Proteins metabolism, Xenopus laevis, Cell Cycle physiology, Oocytes physiology, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-met physiology, Proto-Oncogene Proteins c-raf metabolism
- Abstract
Biological responses of hepatocyte growth factor (HGF) are mediated by the Met receptor tyrosine kinase. Although HGF is a potent mitogen for a variety of cells, the signals required for cell-cycle progression by the Met/HGF receptor are poorly defined. In this study, we have used the Xenopus oocyte system to define the role of various Met proximal-binding partners and downstream signaling pathways in cell-cycle regulation. We show that cell-cycle progression and activation of MAPK and JNK mediated by the oncogenic Met receptor, Tpr-Met, are dependent on its kinase activity and the presence of the twin phosphotyrosine (Y482 & Y489) residues in its C-terminus, but that the recruitment of Grb2 and Shc adaptor proteins is dispensable, implicating other signaling molecules. However, using Met receptor oncoproteins engineered to recruit specific signaling proteins, we demonstrate that recruitment of Grb2 or Shc adaptor proteins is sufficient to induce cell-cycle progression and activation of MAPK and JNK, while the binding of phospholipase-Cgamma or phosphatidylinositol 3-kinase alone fails to elicit these responses. Using various means to block phosphatidylinositol 3-kinase, phospholipase-Cgamma, MEK, JNK, Mos, and Raf1 activity, we show that unlike the fibroblast growth factor receptor, MEK-dependent and independent signaling contribute to Met receptor-mediated cell-cycle progression, but phospholipase-Cgamma or JNK activity and Mos synthesis are not critical. Notably, we demonstrate that Raf1 and phosphatidylinositol 3-kinase signaling are required for cell-cycle progression initiated by the Met receptor, a protein frequently deregulated in human tumors.
- Published
- 2006
- Full Text
- View/download PDF
34. Dishevelled mediates ephrinB1 signalling in the eye field through the planar cell polarity pathway.
- Author
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Lee HS, Bong YS, Moore KB, Soria K, Moody SA, and Daar IO
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Cell Polarity, Dishevelled Proteins, Retina cytology, Retina metabolism, Signal Transduction, Stem Cells metabolism, Xenopus Proteins, Xenopus laevis metabolism, Cell Movement, Ephrin-B1 metabolism, Phosphoproteins metabolism, Retina embryology, Xenopus laevis embryology
- Abstract
An important step in retinal development is the positioning of progenitors within the eye field where they receive the local environmental signals that will direct their ultimate fate. Recent evidence indicates that ephrinB1 functions in retinal progenitor movement, but the signalling pathway is unclear. We present evidence that ephrinB1 signals through its intracellular domain to control retinal progenitor movement into the eye field by interacting with Xenopus Dishevelled (Xdsh), and by using the planar cell polarity (PCP) pathway. Blocking Xdsh translation prevents retinal progeny from entering the eye field, similarly to the morpholino-mediated loss of ephrinB1 (ref. 2). Overexpression of Xdsh can rescue the phenotype induced by loss of ephrinB1, and this rescue (as well as a physical association between Xdsh and ephrinB1) is completely dependent on the DEP (Dishevelled, Egl-10, Pleckstrin) domain of Xdsh. Similar gain- and loss-of-function experiments suggest that Xdsh associates with ephrinB1 and mediates ephrinB1 signalling through downstream members of the PCP pathway during eye field formation.
- Published
- 2006
- Full Text
- View/download PDF
35. KSR regulation of the Raf-MEK-ERK cascade.
- Author
-
Ritt DA, Daar IO, and Morrison DK
- Subjects
- Animals, Extracellular Signal-Regulated MAP Kinases metabolism, Fluorescent Antibody Technique, MAP Kinase Kinase Kinases metabolism, Mice, NIH 3T3 Cells, Oocytes metabolism, Signal Transduction, Xenopus laevis, raf Kinases metabolism, Protein Kinases physiology
- Abstract
Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that functions as a molecular scaffold to enhance signaling between the core kinase components of the ERK cascade--Raf, MEK, and ERK. KSR interacts constitutively with MEK and translocates from the cytosol to the plasma membrane on Ras activation. At the membrane, KSR coordinates the assembly of a multiprotein complex containing Raf, MEK, and ERK and facilitates signal transmission from Raf to MEK and ERK. In this chapter, we will describe methods for assessing KSR function in response to Ras pathway activation. Protocols will be included that examine the ERK scaffolding activity and subcellular localization of KSR.
- Published
- 2006
- Full Text
- View/download PDF
36. Contribution of JNK, Mek, Mos and PI-3K signaling to GVBD in Xenopus oocytes.
- Author
-
Mood K, Bong YS, Lee HS, Ishimura A, and Daar IO
- Subjects
- Animals, Cell Cycle drug effects, Cell Cycle physiology, Cytoplasmic Vesicles drug effects, Cytoplasmic Vesicles metabolism, Enzyme Activation drug effects, Enzyme Activation genetics, Enzyme Inhibitors pharmacology, Gene Expression Regulation, Enzymologic drug effects, Gene Expression Regulation, Enzymologic genetics, MAP Kinase Kinase 4, Oligodeoxyribonucleotides, Antisense pharmacology, Oocytes metabolism, Progesterone pharmacology, Proto-Oncogene Proteins c-mos genetics, Signal Transduction genetics, JNK Mitogen-Activated Protein Kinases, MAP Kinase Kinase Kinases metabolism, Mitogen-Activated Protein Kinase Kinases metabolism, Phosphatidylinositol 3-Kinases metabolism, Proto-Oncogene Proteins c-mos metabolism, Signal Transduction physiology, Xenopus laevis metabolism
- Abstract
In Xenopus oocytes, induction of the G2/M transition by progesterone is a complex process that is promoted by a network of signaling molecules whose cumulative effect results in the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). We examined the role of Mos, Mek, PI-3 kinase and c-Jun N-terminal kinase (JNK) in progesterone stimulation of GVBD. Expression of an activated form of JNK neither induced nor enhanced progesterone-mediated GVBD in oocytes, suggesting a limited role in cell-cycle progression. We blocked Mek, Mos and PI-3 kinase activities by a variety of means that included expression of dominant-negative kinase suppressor of Ras (DnKSR), expression of a dominant-negative PI-3 kinase (DnPI3K), treatment of oocytes with a Mek inhibitor (U1026) or PI-3 kinase (LY294002) inhibitor, and introduction of Mos antisense morpholinos. Inhibition of any one pathway alone failed to block GVBD induced by either high or low concentrations of progesterone. In contrast, inhibiting Mos or Mek function in addition to abrogating PI-3 kinase activity effectively blocked oocyte maturation. Furthermore, by expressing suboptimal amounts of Mos in conjunction with an activated form of Mek and an activated form of the p110 catalytic subunit of PI-3 kinase, we show cooperation among these signaling molecules toward the induction of GVBD. Moreover, expression of optimal amounts of these three proteins in conjunction with inhibitors of Mos, Mek or PI-3 kinase demonstrated that activated Mek-induced GVBD is independent of Mos or PI-3 kinase activity. In addition, Mos-induced GVBD is dependent upon Mek activity, but does not require PI-3 kinase activity. Finally, Mos appears to be a major contributor to GVBD induced by activated PI-3 kinase, while Mek is a minor contributor to this process.
- Published
- 2004
- Full Text
- View/download PDF
37. Ectopic EphA4 receptor induces posterior protrusions via FGF signaling in Xenopus embryos.
- Author
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Park EK, Warner N, Bong YS, Stapleton D, Maeda R, Pawson T, and Daar IO
- Subjects
- Amino Acid Motifs, Animals, Blotting, Western, Catalysis, Cell Adhesion, Cell Membrane metabolism, Cloning, Molecular, Fibroblast Growth Factor 8, In Situ Hybridization, Mice, Mutation, Phenotype, Phosphorylation, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, RNA chemistry, RNA metabolism, RNA, Messenger metabolism, Receptor, EphA4 biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Tyrosine chemistry, Xenopus, src Homology Domains, Fibroblast Growth Factors metabolism, Gene Expression Regulation, Developmental, Receptor, EphA4 physiology
- Abstract
The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.
- Published
- 2004
- Full Text
- View/download PDF
38. Morphogenesis during Xenopus gastrulation requires Wee1-mediated inhibition of cell proliferation.
- Author
-
Murakami MS, Moody SA, Daar IO, and Morrison DK
- Subjects
- Animals, CDC2 Protein Kinase metabolism, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Down-Regulation, Embryo, Nonmammalian metabolism, Mesoderm metabolism, Phosphorylation, Xenopus, Xenopus Proteins, Zygote metabolism, cdc25 Phosphatases genetics, cdc25 Phosphatases metabolism, Cell Division physiology, Gastrula metabolism, Nuclear Proteins, Protein-Tyrosine Kinases metabolism
- Abstract
Major developmental events in early Xenopus embryogenesis coincide with changes in the length and composition of the cell cycle. These changes are mediated in part through the regulation of CyclinB/Cdc2 and they occur at the first mitotic cell cycle, the mid-blastula transition (MBT) and at gastrulation. In this report, we investigate the contribution of maternal Wee1, a kinase inhibitor of CyclinB/Cdc2, to these crucial developmental transitions. By depleting Wee1 protein levels using antisense morpholino oligonucleotides, we show that Wee1 regulates M-phase entry and Cdc2 tyrosine phosphorylation in early gastrula embryos. Moreover, we find that Wee1 is required for key morphogenetic movements involved in gastrulation, but is not needed for the induction of zygotic transcription. In addition, Wee1 is positively regulated by tyrosine autophosphorylation in early gastrula embryos and this upregulation of Wee1 activity is required for normal gastrulation. We also show that overexpression of Cdc25C, a phosphatase that activates the CyclinB/Cdc2 complex, induces gastrulation defects that can be rescued by Wee1, providing additional evidence that cell cycle inhibition is crucial for the gastrulation process. Together, these findings further elucidate the developmental function of Wee1 and demonstrate the importance of cell cycle regulation in vertebrate morphogenesis.
- Published
- 2004
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- View/download PDF
39. Tyr-298 in ephrinB1 is critical for an interaction with the Grb4 adaptor protein.
- Author
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Bong YS, Park YH, Lee HS, Mood K, Ishimura A, and Daar IO
- Subjects
- Adaptor Proteins, Vesicular Transport chemistry, Amino Acid Sequence, Animals, Binding Sites, Cells, Cultured, Ephrin-B1 chemistry, Humans, Molecular Sequence Data, Oncogene Proteins chemistry, Oncogene Proteins metabolism, Phosphorylation, Sequence Alignment, Tyrosine physiology, Xenopus laevis, src Homology Domains, Adaptor Proteins, Signal Transducing, Adaptor Proteins, Vesicular Transport metabolism, Ephrin-B1 metabolism, Xenopus Proteins chemistry, Xenopus Proteins metabolism
- Abstract
The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are thought to play a role in the regulation of cell adhesion and migration during development by mediating cell-to-cell signalling events. The transmembrane ephrinB protein is a bidirectional signalling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase residing on another cell. The reverse signal is transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Previous work from our laboratory has implicated the activated FGFR1 (fibroblast growth factor receptor 1) as a regulator of a de-adhesion signal that results from overexpression of ephrinB1. In the present study, we report the isolation of Xenopus Grb4 (growth-factor-receptor-bound protein 4), an ephrinB1-interacting protein, and we show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4, and the resulting mutants were expressed along with ephrinB1 and an activated FGFR in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the Src homology 2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, whereas Tyr-305 and Tyr-310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine-phosphorylation-dependent event.
- Published
- 2004
- Full Text
- View/download PDF
40. Morphogenetic movements underlying eye field formation require interactions between the FGF and ephrinB1 signaling pathways.
- Author
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Moore KB, Mood K, Daar IO, and Moody SA
- Subjects
- Animals, Body Patterning physiology, Cell Lineage physiology, Embryo, Nonmammalian cytology, Embryo, Nonmammalian metabolism, Gastrula cytology, Gastrula metabolism, Gene Expression Regulation, Developmental physiology, Genes, Regulator physiology, Morphogenesis physiology, Phenotype, Retina cytology, Retina metabolism, Signal Transduction physiology, Stem Cells cytology, Stem Cells metabolism, Xenopus laevis metabolism, Cell Movement physiology, Embryo, Nonmammalian embryology, Ephrin-B1 metabolism, Fibroblast Growth Factors metabolism, Retina embryology, Xenopus laevis embryology
- Abstract
The definitive retinal progenitors of the eye field are specified by transcription factors that both promote a retinal fate and control cell movements that are critical for eye field formation. However, the molecular signaling pathways that regulate these movements are largely undefined. We demonstrate that both the FGF and ephrin pathways impact eye field formation. Activating the FGF pathway before gastrulation represses cellular movements in the presumptive anterior neural plate and prevents cells from expressing a retinal fate, independent of mesoderm induction or anterior-posterior patterning. Inhibiting the FGF pathway promotes cell dispersal and significantly increases eye field contribution. ephrinB1 reverse signaling is required to promote cellular movements into the eye field, and can rescue the FGF receptor-induced repression of retinal fate. These results indicate that FGF modulation of ephrin signaling regulates the positioning of retinal progenitor cells within the definitive eye field.
- Published
- 2004
- Full Text
- View/download PDF
41. Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos.
- Author
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Iraha F, Saito Y, Yoshida K, Kawakami M, Izutsu Y, Daar IO, and Maéno M
- Subjects
- Animals, Biomarkers, Blood Cells cytology, Blood Vessels cytology, Blood Vessels physiology, Fibroblast Growth Factors physiology, Globins genetics, In Situ Hybridization, Receptor Protein-Tyrosine Kinases genetics, Receptor, TIE-2, Signal Transduction physiology, Xenopus laevis, Blood Cells physiology, Blood Vessels embryology, Cell Differentiation physiology, Cell Lineage physiology, Embryo, Nonmammalian physiology
- Abstract
In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie-2, a Xenopus homolog of the tie-2 receptor tyrosine kinase, was examined. Whole-mount in situ hybridization analysis revealed that Xtie-2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie-2-positive cells are predominantly present just outside the boundary of alpha-globin-positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie-2 and alpha-globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a "dorsal determinant" from the vegetal cytoplasm at the 1-cell stage. Second, expression of both Xtie-2 and alpha-globin were markedly induced in ectodermal explants (animal caps) from embryos co-injected with activin and bone morphogenetic protein (BMP)-4 RNA. Furthermore, both Xtie-2 and alpha-globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP-4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP-4-dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages.
- Published
- 2002
- Full Text
- View/download PDF
42. SNT1/FRS2 mediates germinal vesicle breakdown induced by an activated FGF receptor1 in Xenopus oocytes.
- Author
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Mood K, Friesel R, and Daar IO
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Binding Sites, Blotting, Western, Cell Cycle, Enzyme Inhibitors pharmacology, Histones metabolism, Intracellular Signaling Peptides and Proteins, Ligands, MAP Kinase Signaling System, Mutation, Phosphorylation, Plasmids metabolism, Precipitin Tests, Progesterone metabolism, Protein Binding, Protein Kinases metabolism, Proteins metabolism, RNA metabolism, Receptor, Fibroblast Growth Factor, Type 1, Xenopus, Germ Cells metabolism, Membrane Proteins metabolism, Oocytes metabolism, Phosphoproteins metabolism, Receptor Protein-Tyrosine Kinases metabolism, Receptors, Fibroblast Growth Factor metabolism
- Abstract
The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from the fibroblast growth factor receptor (FGFR), which plays vital roles during embryogenesis. Activating FGFR mutations cause several craniosynostoses and dwarfism syndromes in humans. Here we show that the Xenopus homolog of mammalian FRS-2 (XFRS2) is essential for the induction of oocyte maturation by an XFGFR1 harboring an activating mutation (XFGFR1act). Using a dominant-negative form of kinase suppressor of Ras, we show the Mek activity is required for germinal vesicle breakdown (GVBD) induced by co-expression of XFGFR1act and XFRS2, but this activity is not required for progesterone-induced GVBD. Furthermore, Mek/MAPK activity is critical for the induction and/or maintenance of H1 kinase activity at metaphase of meiosis II in progesterone-treated oocytes. An activated XFGFR1 containing a mutation in the phospholipase Cgamma binding site (XFGFR1actY672F) displayed a reduced ability to induce cell-cycle progression in oocytes, suggesting phospholipase Cgamma may not be necessary but that it augments XFGFR signaling in this system. Oocytes co-expressing XFGFR1act and XFRS2 showed substantial H1 kinase activity, but this activity was blocked when the oocytes were treated with the phosphatidylinositol 3-kinase inhibitor LY294002. Although phosphatidylinositol 3-kinase activity is essential for XFGFR1act/XFRS2-induced oocyte maturation, this activity is not required for maturation induced by progesterone. Finally, ectopic expression of Xspry2, a negative regulator of XFGFR signaling, greatly reduced MAPK activation and GVBD induced by the expression of either XFGFR1act plus XFRS2 or activated Ras (H-RasV12). In contrast, Xspry2 did not prevent GVBD induced by an activated form of Raf1, suggesting that Xspry2 exerts its inhibitory function upstream or parallel to Raf and downstream of Ras.
- Published
- 2002
- Full Text
- View/download PDF
43. Low-molecular-weight protein tyrosine phosphatase is a positive component of the fibroblast growth factor receptor signaling pathway.
- Author
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Park EK, Warner N, Mood K, Pawson T, and Daar IO
- Subjects
- Amino Acid Sequence, Animals, In Situ Hybridization, Mitogen-Activated Protein Kinases metabolism, Molecular Sequence Data, Oligonucleotides, Antisense genetics, Oocytes physiology, Protein Isoforms, Protein Tyrosine Phosphatase, Non-Receptor Type 1, Protein Tyrosine Phosphatases chemistry, Protein Tyrosine Phosphatases genetics, Protein Tyrosine Phosphatases isolation & purification, Receptor, Fibroblast Growth Factor, Type 1, Sequence Alignment, Xenopus Proteins genetics, Embryo, Nonmammalian physiology, Oligonucleotides, Antisense metabolism, Protein Tyrosine Phosphatases metabolism, Receptor Protein-Tyrosine Kinases metabolism, Receptors, Fibroblast Growth Factor metabolism, Signal Transduction physiology, Xenopus Proteins metabolism, Xenopus laevis embryology
- Abstract
Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) has been implicated in the regulation of cell growth and actin rearrangement mediated by several receptor tyrosine kinases, including platelet-derived growth factor and epidermal growth factor. Here we identify the Xenopus laevis homolog of LMW-PTP1 (XLPTP1) as an additional positive regulator in the fibroblast growth factor (FGF) signaling pathway during Xenopus development. XLPTP1 has an expression pattern that displays substantial overlap with FGF receptor 1 (FGFR1) during Xenopus development. Using morpholino antisense technology, we show that inhibition of endogenous XLPTP1 expression dramatically restricts anterior and posterior structure development and inhibits mesoderm formation. In ectodermal explants, loss of XLPTP1 expression dramatically blocks the induction of the early mesoderm gene, Xbrachyury (Xbra), by FGF and partially blocks Xbra induction by Activin. Moreover, FGF-induced activation of mitogen-activated protein (MAP) kinase is also inhibited by XLPTP1 morpholino antisense oligonucleotides; however, introduction of RNA encoding XLPTP1 is able to rescue morphological and biochemical effects of antisense inhibition. Inhibition of FGF-induced MAP kinase activity due to loss of XLPTP1 is also rescued by an active Ras, implying that XLPTP1 may act upstream of or parallel to Ras. Finally, XLPTP1 physically associates only with an activated FGFR1, and this interaction requires the presence of SNT1/FRS-2 (FGFR substrate 2). Although LMW-PTP1 has been shown to participate in other receptor systems, the data presented here also reveal XLPTP1 as a new and important component of the FGF signaling pathway.
- Published
- 2002
- Full Text
- View/download PDF
44. Xpbx1b and Xmeis1b play a collaborative role in hindbrain and neural crest gene expression in Xenopus embryos.
- Author
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Maeda R, Ishimura A, Mood K, Park EK, Buchberg AM, and Daar IO
- Subjects
- Amino Acid Sequence, Animals, Blotting, Northern, Blotting, Western, DNA, Complementary metabolism, DNA-Binding Proteins biosynthesis, Dimerization, Gene Library, Homeodomain Proteins biosynthesis, Immunohistochemistry, In Situ Hybridization, Molecular Sequence Data, Myeloid Ecotropic Viral Integration Site 1 Protein, Neoplasm Proteins biosynthesis, Pre-B-Cell Leukemia Transcription Factor 1, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Proto-Oncogene Proteins biosynthesis, RNA metabolism, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Transcription, Genetic, Xenopus Proteins biosynthesis, DNA-Binding Proteins physiology, Homeodomain Proteins physiology, Neoplasm Proteins physiology, Neural Crest metabolism, Proto-Oncogene Proteins physiology, Xenopus embryology, Xenopus Proteins physiology
- Abstract
Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development.
- Published
- 2002
- Full Text
- View/download PDF
45. Docking protein SNT1 is a critical mediator of fibroblast growth factor signaling during Xenopus embryonic development.
- Author
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Akagi K, Kyun Park E, Mood K, and Daar IO
- Subjects
- Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Embryo, Nonmammalian metabolism, Embryonic Development, Gene Expression Regulation, Developmental, In Situ Hybridization, Membrane Proteins biosynthesis, Membrane Proteins genetics, Mesoderm metabolism, Molecular Sequence Data, Morphogenesis, Oligodeoxyribonucleotides, Antisense pharmacology, Phosphoproteins biosynthesis, Phosphoproteins genetics, Receptor Protein-Tyrosine Kinases biosynthesis, Receptor Protein-Tyrosine Kinases genetics, Receptor, Fibroblast Growth Factor, Type 1, Receptors, Fibroblast Growth Factor biosynthesis, Receptors, Fibroblast Growth Factor genetics, Sequence Alignment, Sequence Homology, Amino Acid, Species Specificity, Vertebrates genetics, Xenopus Proteins biosynthesis, Xenopus Proteins genetics, Xenopus laevis genetics, Xenopus laevis metabolism, Fibroblast Growth Factor 1 physiology, MAP Kinase Signaling System, Membrane Proteins physiology, Phosphoproteins physiology, Receptor Protein-Tyrosine Kinases physiology, Receptors, Fibroblast Growth Factor physiology, Xenopus Proteins physiology, Xenopus laevis embryology
- Abstract
The docking protein SNT1/FRS2 (fibroblast growth factor receptor substrate 2) is implicated in the transmission of extracellular signals from several growth factor receptors to the mitogen-activated protein (MAP) kinase signaling cascade, but its biological function during development is not well characterized. Here, we show that the Xenopus homolog of mammalian SNT1/FRS-2 (XSNT1) plays a critical role in the appropriate formation of mesoderm-derived tissue during embryogenesis. XSNT1 has an expression pattern that is quite similar to the fibroblast growth factor receptor-1 (FGFR1) during Xenopus development. Ectopic expression of XSNT1 markedly enhanced the embryonic defects induced by an activated FGF receptor, and increased the MAP kinase activity as well as the expression of a mesodermal marker in response to FGF receptor signaling. A loss-of-function study using antisense XSNT1 morpholino oligonucleotides (XSNT-AS) shows severe malformation of trunk and posterior structures. Moreover, XSNT-AS disrupts muscle and notochord formation, and inhibits FGFR-induced MAP kinase activation. In ectodermal explants, XSNT-AS blocks FGFR-mediated induction of mesoderm and the accompanying elongation movements. Our results indicate that XSNT1 is a critical mediator of FGF signaling and is required for early Xenopus development., (Copyright 2002 Wiley-Liss, Inc.)
- Published
- 2002
- Full Text
- View/download PDF
46. Xmeis1, a protooncogene involved in specifying neural crest cell fate in Xenopus embryos.
- Author
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Maeda R, Mood K, Jones TL, Aruga J, Buchberg AM, and Daar IO
- Subjects
- Alternative Splicing, Animals, Antigens, Differentiation, Ectoderm transplantation, Embryo, Nonmammalian, Embryonic Induction, Homeodomain Proteins genetics, Mesoderm, Myeloid Ecotropic Viral Integration Site 1 Protein, Neoplasm Proteins genetics, Oncogene Proteins, Protein Binding, RNA, Messenger metabolism, Trans-Activators, Transcription Factors, Xenopus, Zinc Finger Protein GLI1, Homeodomain Proteins metabolism, Neoplasm Proteins metabolism, Nervous System embryology, Neural Crest embryology, Proto-Oncogenes, Xenopus Proteins
- Abstract
Meis1 (Myeloid Ecotropic viral Integration Site 1) is a homeobox gene that was originally isolated as a common site of viral integration in myeloid tumors of the BXH-2 recombinant inbred mice strain. We previously isolated a Xenopus homolog of Meis1 (Xmeis1). Here we show that Xmeis1 may play a significant role in neural crest development. In developing Xenopus embryos, Xmeis1 displays a broad expression pattern, but strong expression is observed in tissue of neural cell fate, such as midbrain, hindbrain, the dorsal portion of the neural tube, and neural crest derived branchial arches. In animal cap explants, overexpression of Xmeis1b, an alternatively spliced form of Xmeis1, induces expression of neural crest marker genes in the absence of mesoderm. Moreover, Xmeis1b induces XGli-3 and XZic3, pre-pattern genes involved at the earliest stages of neural crest development, and like these two genes, can induce ectopic pigmented cell masses when overexpressed in developing embryos. Misexpression of Xmeis1b also induces ectopic expression of neural crest markers along the antero-posterior axis of the neural tube in developing Xenopus embryos. In contrast, Xmeis1a, another splice variant, is much less effective at inducing these effects. These data suggest that Xmeis1b is involved in neural crest cell fate specification during embryogenesis, and can functionally intersect with the Gli/Zic signal transduction pathway.
- Published
- 2001
- Full Text
- View/download PDF
47. Cloning protein tyrosine kinases by screening cDNA libraries with antiphosphotyrosine antibodies.
- Author
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Chong LD and Daar IO
- Subjects
- Antibodies, Cloning, Molecular methods, DNA, Complementary, Escherichia coli genetics, Indicators and Reagents, Phosphotyrosine immunology, Protein-Tyrosine Kinases chemistry, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Gene Library, Phosphotyrosine analysis, Protein-Tyrosine Kinases genetics, Protein-Tyrosine Kinases metabolism
- Published
- 2001
- Full Text
- View/download PDF
48. Involvement of BMP-4/msx-1 and FGF pathways in neural induction in the Xenopus embryo.
- Author
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Ishimura A, Maeda R, Takeda M, Kikkawa M, Daar IO, and Maéno M
- Subjects
- Animals, Body Patterning, Bone Morphogenetic Protein 4, Carrier Proteins, Cell Differentiation, Cell Lineage, DNA Primers chemistry, Ectoderm physiology, Enzyme Inhibitors pharmacology, Etoposide metabolism, In Situ Hybridization, MSX1 Transcription Factor, Morphogenesis, Mutation, Nervous System metabolism, Proteins metabolism, RNA, Messenger analysis, RNA, Messenger genetics, Recombinant Fusion Proteins metabolism, Reverse Transcriptase Polymerase Chain Reaction, Xenopus Proteins, Xenopus laevis physiology, Bone Morphogenetic Proteins physiology, Embryo, Nonmammalian innervation, Embryonic Induction physiology, Fibroblast Growth Factors physiology, Homeodomain Proteins physiology, Nervous System embryology, Signal Transduction physiology, Transcription Factors, Xenopus laevis embryology
- Abstract
The msx homeodomain protein is a downstream transcription factor of the bone morphogenetic protein (BMP)-4 signal and a key regulator for neural tissue differentiation. Xmsx-1 antagonizes the dorsal expression of noggin and cerberus, as revealed by in situ hybridization and reverse transcription-polymerase chain reaction assays. In animal cap explants, Xmsx-1 and BMP-4 inhibit the neural tissue differentiation induced by noggin or cerberus. A loss-of-function study using the Xmsx-1/VP-16 fusion construct indicated that neural tissue formation was directly induced by the injection of fusion ribonucleic acid, although the expression of neural cell adhesion molecule (N-CAM) in the cap was less than that in the cap injected with tBR or noggin. In contrast to the single cap assay, unexpectedly, both BMP-4 and Xmsx-1 failed to inhibit neurulation in the ectodermal explants to which the organizer mesoderm was attached. The results of cell-lineage tracing experiments indicated that the neural cells were differentiated from the animal pole tissue where the excess RNA of either BMP-4 or Xmsx-1 was injected, whereas notochord was differentiated from the organizer mesoderm. Neural tissue differentiated from BMP-4-injected ectodermal cells strongly expressed posterior neural markers, such as hoxB9 and krox20, suggesting that the posterior neural cells differentiated regardless of the existence of the BMP signal. The introduction of a dominant-negative form of the fibroblast growth factor (FGF) receptor (XFD) into the ectodermal cells drastically reduced the expression of pan and posterior neural markers (N-CAM and hoxB-9) if co-injected with BMP-4 RNA, although XFD alone at the same dose did not shut down the expression of N-CAM in the combination explants. Therefore, it is proposed that an FGF-related molecule was involved in the direct induction of posterior neural tissue in the inducing signals from the organizer mesoderm in vivo.
- Published
- 2000
- Full Text
- View/download PDF
49. Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos.
- Author
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Chong LD, Park EK, Latimer E, Friesel R, and Daar IO
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Cell Adhesion drug effects, Chick Embryo, Conserved Sequence, Embryo, Nonmammalian drug effects, Embryo, Nonmammalian metabolism, Ephrin-B1, Fibroblast Growth Factors pharmacology, Gene Expression, Membrane Proteins chemistry, Membrane Proteins genetics, Mitogen-Activated Protein Kinases metabolism, Molecular Sequence Data, Mutation genetics, Phosphorylation drug effects, Phosphotyrosine metabolism, Protein Binding, Receptors, Fibroblast Growth Factor genetics, Receptors, Platelet-Derived Growth Factor physiology, Retina drug effects, Retina embryology, Retina metabolism, Signal Transduction drug effects, Xenopus laevis embryology, Embryo, Nonmammalian cytology, Membrane Proteins metabolism, Receptor Cross-Talk drug effects, Receptors, Fibroblast Growth Factor metabolism
- Abstract
The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. Genetic evidence suggests that ephrins may transduce signals and become tyrosine phosphorylated during embryogenesis. However, the induction and functional significance of ephrin phosphorylation is not yet clear. Here, we report that when we used ectopically expressed proteins, we found that an activated fibroblast growth factor (FGF) receptor associated with and induced the phosphorylation of ephrin B1 on tyrosine. Moreover, this phosphorylation reduced the ability of overexpressed ephrin B1 to reduce cell adhesion. In addition, we identified a region in the cytoplasmic tail of ephrin B1 that is critical for interaction with the FGF receptor; we also report FGF-induced phosphorylation of ephrins in a neural tissue. This is the first demonstration of communication between the FGF receptor family and the Eph ligand family and implicates cross talk between these two cell surface molecules in regulating cell adhesion.
- Published
- 2000
- Full Text
- View/download PDF
50. Xenopus CRMP-2 is an early response gene to neural induction.
- Author
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Kamata T, Daar IO, Subleski M, Copeland T, Kung HF, and Xu RH
- Subjects
- Amino Acid Sequence, Animals, Bone Morphogenetic Proteins biosynthesis, Embryo, Nonmammalian metabolism, Gastrula metabolism, In Situ Hybridization, Intercellular Signaling Peptides and Proteins, Molecular Sequence Data, Nervous System metabolism, Polymerase Chain Reaction methods, Semaphorin-3A, Sequence Homology, Amino Acid, Signal Transduction physiology, Transcription, Genetic, Xenopus, Embryonic Development, Gene Expression Regulation, Developmental physiology, Nerve Tissue Proteins genetics, Nervous System embryology
- Abstract
A neural specific protein, CRMP-2 (for Collapsin Response Mediator Protein-2), is considered to mediate collapsin-induced growth cone collapse during neural development. We have isolated the Xenopus homologue of the CRMP-2 (XCRMP-2) cDNA and studied the expression of XCRMP-2 mRNA and protein during neural induction. Induction of XCRMP-2 mRNA and protein expression, like N-CAM, occurred at the midgastrula stage and increased through early neural developmental stages. Whole mount in situ hybridization demonstrated that expression of XCRMP-2 mRNA was localized in neural tissues such as the neural plate and tube at early stages, while its expression in the brain, spinal cord, and eyes was observed at later stages. Immunostaining of Xenopus embryos with the antibody against CRMP-2 also showed that the protein was specifically expressed in the neural tissues at early stages. XCRMP-2 expression was induced by neural inducers such as noggin and chordin which antagonize a neural inhibitor, BMP4. A dominant negative BMP receptor also induced XCRMP-2 expression, suggesting that transcription of XCRMP-2 gene was negatively regulated by the BMP4 signaling. These results indicate that expression of XCRMP-2 is an early response marking neural commitment, and that transcriptional control of XCRMP-2 gene, is one of the targets of BMP4 signaling.
- Published
- 1998
- Full Text
- View/download PDF
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