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ABSTRACT: Evolutionarily conserved Tcf/Lef transcription factors (Lef1, Tcf7, Tcf7l1, and Tcf7l2) mediate gene expression regulated by Wnt/β-catenin signaling, which has multiple roles in early embryogenesis, organogenesis, adult tissue homeostasis, and tissue regeneration. However, the spatiotemporal dynamics of Tcf/Lef activity during these events remain poorly understood. We generated stable transgenic zebrafish lines carrying a new Wnt/β-catenin signaling reporter, Tcf/Lef-miniP:dGFP. The reporter revealed the transcriptional activities of four Tcf/Lef members controlled by Wnt/β-catenin signaling, which were expressed in known Wnt/β-catenin signaling-active sites during embryogenesis, organ development and growth, and tissue regeneration. We used the transgenic lines to demonstrate the contribution of Tcf/Lef-mediated Wnt/β-catenin signaling to the development of the anterior lateral line, dorsal and secondary posterior lateral lines, and gill filaments. Thus, these reporter lines are highly useful tools for studying Tcf/Lef-mediated Wnt/β-catenin signaling-dependent processes.
Developmental Biology 07/2012; 370(1):71-85. · 4.07 Impact Factor
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ABSTRACT: Nemo-like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/β-catenin signalling. However, the roles of NLK in Wnt/β-catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor-1 (Lef1)-mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC-like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/β-catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/β-catenin signalling pathway.
The EMBO Journal 02/2012; 31(8):1904-15. · 9.20 Impact Factor
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ABSTRACT: Spatially and temporally controlled gene expression, including transcription, several mRNA processing steps, and the export of mature mRNA to the cytoplasm, is essential for developmental processes. It is well known that RNA helicases of the DExD/H-box protein family are involved in these gene expression processes, including transcription, pre-mRNA splicing, and rRNA biogenesis. Although one DExD/H-box protein, Prp5, a homologue of vertebrate Ddx46, has been shown to play important roles in pre-mRNA splicing in yeast, the in vivo function of Ddx46 remains to be fully elucidated in metazoans. In this study, we isolated zebrafish morendo (mor), a mutant that shows developmental defects in the digestive organs and brain, and found that it encodes Ddx46. The Ddx46 transcript is maternally supplied, and as development proceeds in zebrafish larvae, its ubiquitous expression gradually becomes restricted to those organs. The results of whole-mount in situ hybridization showed that the expression of various molecular markers in these organs is considerably reduced in the Ddx46 mutant. Furthermore, splicing status analysis with RT-PCR revealed unspliced forms of mRNAs in the digestive organ and brain tissues of the Ddx46 mutant, suggesting that Ddx46 may be required for pre-mRNA splicing during zebrafish development. Therefore, our results suggest a model in which zebrafish Ddx46 is required for the development of the digestive organs and brain, possibly through the control of pre-mRNA splicing.
PLoS ONE 01/2012; 7(3):e33675. · 4.09 Impact Factor
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ABSTRACT: Although recent findings showed that some Drosophila doublesex and Caenorhabditis elegans mab-3 related genes are expressed in neural tissues during development, their functions have not been fully elucidated. Here, we isolated a zebrafish mutant, ha2, that shows defects in telencephalic neurogenesis and found that ha2 encodes Doublesex and MAB-3 related transcription factor like family A2 (Dmrta2). dmrta2 expression is restricted to the telencephalon, diencephalon and olfactory placode during somitogenesis. We found that the expression of the proneural gene, neurogenin1, in the posterior and dorsal region of telencephalon (posterior-dorsal telencephalon) is markedly reduced in this mutant at the 14-somite stage without any defects in cell proliferation or cell death. In contrast, the telencephalic expression of her6, a Hes-related gene that is known to encode a negative regulator of neurogenin1, expands dramatically in the ha2 mutant. Based on over-expression experiments and epistatic analyses, we propose that zebrafish Dmrta2 controls neurogenin1 expression by repressing her6 in the posterior-dorsal telencephalon. Furthermore, the expression domains of the telencephalic marker genes, foxg1 and emx3, and the neuronal differentiation gene, neurod, are downregulated in the ha2 posterior-dorsal telencephalon during somitogenesis. These results suggest that Dmrta2 plays important roles in the specification of the posterior-dorsal telencephalic cell fate during somitogenesis.
Genes to Cells 11/2011; 16(11):1097-109. · 2.68 Impact Factor
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ABSTRACT: Nemo-like kinase (NLK) is an evolutionarily conserved protein kinase that phosphorylates several transcription factors. However, the molecular mechanisms that regulate NLK activity have been poorly understood. Here we show that homodimerization of NLK is required for its activation and nuclear localization. Biochemical analysis revealed that NLK is activated through intermolecular autophosphorylation of NLK dimers at Thr-286. Mutation of NLK at Cys-425, which corresponds to the defect in the Caenorhabditis elegans NLK homologue lit-1, prevented NLK dimerization, rendering NLK defective in both nuclear localization and kinase activity. By contrast, the external addition of nerve growth factor, which has been previously identified as an NLK activator, induced dimerization and Thr-286 autophosphorylation of endogenous NLK proteins. In addition, both dimerization and Thr-286 phosphorylation of NLK were found to be essential for induction of neurite-like cellular processes by NLK. The present findings suggest that dimerization is an initial key event required for the functional activation of NLK.
Molecular biology of the cell 01/2011; 22(2):266-77. · 5.98 Impact Factor
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Mai Yamamoto,
Ryoko Morita,
Takamasa Mizoguchi,
Hiromi Matsuo,
Miho Isoda, Tohru Ishitani,
Ajay B Chitnis,
Kunihiro Matsumoto,
J Gage Crump,
Katsuto Hozumi,
Shigenobu Yonemura,
Koichi Kawakami,
Motoyuki Itoh
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ABSTRACT: In the developing embryo, cell-cell signalling is necessary for tissue patterning and structural organization. During midline development, the notochord plays roles in the patterning of its surrounding tissues while forming the axial structure; however, how these patterning and structural roles are coordinated remains elusive. Here, we identify a mechanism by which Notch signalling regulates the patterning activities and structural integrity of the notochord. We found that Mind bomb (Mib) ubiquitylates Jagged 1 (Jag1) and is essential in the signal-emitting cells for Jag1 to activate Notch signalling. In zebrafish, loss- and gain-of-function analyses showed that Mib-Jag1-Notch signalling favours the development of non-vacuolated cells at the expense of vacuolated cells in the notochord. This leads to changes in the peri-notochordal basement membrane formation and patterning surrounding the muscle pioneer cells. These data reveal a previously unrecognized mechanism regulating the patterning and structural roles of the notochord by Mib-Jag1-Notch signalling-mediated cell-fate determination.
Development 08/2010; 137(15):2527-37. · 6.60 Impact Factor
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Developmental Biology 08/2010; 344(1):497-498. · 4.07 Impact Factor
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ABSTRACT: Delta family proteins are transmembrane molecules that bind Notch receptors and activate downstream signaling events in neighboring cells. In addition to serving as Notch ligands, Notch-independent roles for Delta have been suggested but are not fully understood. Here, we demonstrate a previously unrecognized role for Delta in filopodial actin formation. Delta1 and Delta4, but not Delta3, exhibit filopodial protrusive activity, and this activity is independent of Notch signaling. The filopodial activity of Delta1 does not depend on the PDZ-binding domain at the C-terminus; however, the intracellular membrane-proximal region that is anchored to the plasma membrane plays an important role in filopodial activity. We further identified a Notch-independent role of DeltaD in neuronal cell migration in zebrafish. These findings suggest a possible functional link between Notch-independent filopodial activity of Delta and the control of cell motility.
Biochemical and Biophysical Research Communications 07/2010; 398(1):118-24. · 2.48 Impact Factor
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ABSTRACT: The Notch signalling pathway has a crucial function in determining cell fates in multiple tissues within metazoan organisms. On binding to ligands, the Notch receptor is cleaved proteolytically and releases its intracellular domain (NotchICD). The NotchICD enters the nucleus and acts cooperatively with other factors to stimulate the transcription of target genes. High levels of Notch-mediated transcriptional activation require the formation of a ternary complex consisting of NotchICD, CSL (CBF-1, suppressor of hairless, LAG-1) and a Mastermind family member. However, it is still not clear how the formation of the ternary complex is regulated. Here we show that Nemo-like kinase (NLK) negatively regulates Notch-dependent transcriptional activation by decreasing the formation of this ternary complex. Using a biochemical screen, we identified Notch as a new substrate of NLK. NLK-phosphorylated Notch1ICD is impaired in its ability to form a transcriptionally active ternary complex. Furthermore, knockdown of NLK leads to hyperactivation of Notch signalling and consequently decreases neurogenesis in zebrafish. Our results both define a new function for NLK and reveal a previously unidentified mode of regulation in the Notch signalling pathway.
Nature Cell Biology 03/2010; 12(3):278-85. · 19.49 Impact Factor
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ABSTRACT: Methylation of histone H3 Lys 9 and Lys 27 (H3K9 and H3K27) is associated with transcriptional silencing. Here we show that KDM7, a JmjC domain-containing protein, catalyzes demethylation of both mono- or dimethylated H3K9 and H3K27. Inhibition of KDM7 orthologs in zebrafish resulted in developmental brain defects. KDM7 interacts with the follistatin gene locus, and KDM7 depletion in mammalian neuronal cells suppressed follistatin gene transcription in association with increased levels of dimethylated H3K9 and H3K27. Our findings identify KDM7 as a dual demethylase for H3K9 and H3K27 that functions as an eraser of silencing marks on chromatin during brain development.
Genes & development 03/2010; 24(5):432-7. · 12.08 Impact Factor
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ABSTRACT: Nemo-like kinase (NLK) is known to function as a mitogen-activated protein kinase (MAPK)-like kinase. However, the upstream molecules and molecular mechanisms that regulate NLK activity remain unclear. In the present study, we identified p38 MAPK as an upstream kinase and activator of NLK. p38 regulates the function of NLK via phosphorylation, and this modification can be abrogated by depletion of endogenous p38. In Xenopus laevis embryos, depletion of either p38beta or NLK by antisense morpholino oligonucleotides results in a severe defect in anterior development and impaired expression of endogenous anterior markers. It is notable that morphants of Xenopus p38alpha, another isoform of the p38 MAPK family, exhibited no obvious defects in anterior development. Defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous p38beta expression could be rescued by expression of wild-type NLK but not by expression of mutant NLK lacking the p38beta phosphorylation site. In contrast, defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous NLK expression could not be rescued by expression of p38. These results provide the first evidence that p38 specifically regulates NLK function, which is required for anterior formation in Xenopus development.
Molecular and cellular biology 11/2009; 30(3):675-83. · 6.06 Impact Factor
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ABSTRACT: Nerve growth factor (NGF) promotes neurite outgrowth through regulating cytoskeletal organization and cell adhesion. These activities are modulated by protein phosphorylation. Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-like kinase that phosphorylates several transcription factors. Although NLK is known to be expressed at relatively high levels in the nervous system, its function is not well understood. We found that NGF promotes the translocation of NLK to PC12 cells' leading edges, and triggers NLK kinase activity in them. Activated NLK directly phosphorylates microtubule-associated protein-1B (MAP1B) and the focal adhesion adaptor protein, paxillin. Knockdown of NLK attenuates the phosphorylation of both paxillin and MAP1B and inhibits both the NGF-induced re-distribution of F-actin and neurite outgrowth. We also discovered that NLK is a LiCl-sensitive kinase. LiCl is known to block NGF-induced neurite outgrowth and the phosphorylation of MAP1B and paxillin in PC12 cells. Therefore, the effects of LiCl are mediated in part by blocking NLK activity. These results suggest that NLK controls the dynamics of the cytoskeleton downstream of NGF signaling.
Journal of Neurochemistry 10/2009; 111(5):1104-18. · 4.06 Impact Factor
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ABSTRACT: Nrarp (Notch-regulated ankyrin repeat protein) is a small protein that has two ankyrin repeats. Although Nrarp is known to be an inhibitory component of the Notch signalling pathway that operates in different developmental processes, the in vivo roles of Nrarp have not been fully characterized. Here, we show that Nrarp is a positive regulator in the Wnt signalling pathway. In zebrafish, knockdown of Nrarp-a expression by an antisense morpholino oligonucleotide (MO) results in altered Wnt-signalling-dependent neural-crest-cell development. Nrarp stabilizes LEF1 protein, a pivotal transcription factor in the Wnt signalling cascade, by blocking LEF1 ubiquitination. In accordance with this, the knockdown phenotype of lef1 is similar to that of nrarp-a, at least in part, in its effect on the development of multiple tissues in zebrafish. Furthermore, activation of LEF1 does not affect Notch activity or vice versa. These findings reveal that Nrarp independently regulates canonical Wnt and Notch signalling by modulating LEF1 and Notch protein turnover, respectively.
Nature Cell Biology 12/2005; 7(11):1106-12. · 19.49 Impact Factor
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ABSTRACT: Signal transducer and activator of transcription 3 (STAT3) is activated by the IL-6 family of cytokines and growth factors. STAT3 requires phosphorylation on Ser-727, in addition to tyrosine phosphorylation on Tyr-705, to be transcriptionally active. In IL-6 signaling, the two major pathways that derive from the YXXQ and the YSTV motifs of gp130 cause Ser-727 phosphorylation. Here, we show that TGF-beta-activated kinase 1 (TAK1) interacts with STAT3, that the TAK1-Nemo-like kinase (NLK) pathway is efficiently activated by IL-6 through the YXXQ motif, and that this is the YXXQ-mediated H7-sensitive pathway that leads to STAT3 Ser-727 phosphorylation. Because NLK was recently shown to interact with STAT3, we explored the role of STAT3 in activating this pathway. Depletion of STAT3 diminished the IL-6-induced NLK activation by >80% without inhibiting IL-6-induced TAK1 activation or its nuclear entry. We found that STAT3 functioned as a scaffold for TAK1 and NLK in vivo through a region in its carboxyl terminus. Furthermore, the expression of the STAT3(534-770) region in the nuclei of STAT3-knockdown cells enhanced the IL-6-induced NLK activation in a dose-dependent manner but not the TGFbeta-induced NLK activation. TGFbeta did not cause STAT3 Ser-727 phosphorylation, even when the carboxyl region of STAT3 was expressed in the nuclei. Together, these results indicate that STAT3 enhances the efficiency of its own Ser-727 phosphorylation by acting as a scaffold for the TAK1-NLK kinases, specifically in the YXXQ motif-derived pathway.
Proceedings of the National Academy of Sciences 03/2005; 102(12):4524-9. · 9.68 Impact Factor
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Chie Kanei-Ishii,
Jun Ninomiya-Tsuji,
Jun Tanikawa,
Teruaki Nomura, Tohru Ishitani,
Satoshi Kishida,
Kenji Kokura,
Toshihiro Kurahashi,
Emi Ichikawa-Iwata,
Yongsok Kim,
Kunihiro Matsumoto,
Shunsuke Ishii
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ABSTRACT: The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. Here we report that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps.
Genes & Development 05/2004; 18(7):816-29. · 11.66 Impact Factor
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ABSTRACT: The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.
The EMBO Journal 01/2004; 22(23):6277-88. · 9.20 Impact Factor
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ABSTRACT: The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. Previously, we have shown that a specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. In this study, we investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. We found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.
Molecular and Cellular Biology 03/2003; 23(4):1379-89. · 5.53 Impact Factor
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ABSTRACT: Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Here we show that activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor (beta(2)AR-Rfz-2) containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor (beta(2)AR) and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling.
Molecular and Cellular Biology 02/2003; 23(1):131-9. · 5.53 Impact Factor
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ABSTRACT: Wnt signaling controls a variety of developmental processes. The canonical Wnt/-catenin pathway functions to stabilize -catenin, and the noncanonical Wnt/Ca2 pathway activates Ca2/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca2 pathway activated by Wnt-5a antagonizes the Wnt/-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/-catenin signaling pathway. Here we show that activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overex- pression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor (2AR-Rfz-2) containing the ligand-binding and transmembrane segments from the 2-adrenergic receptor (2AR) and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the -adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits -catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca2 pathway and antagonizes canonical Wnt/-catenin signaling.
Molecular and Cellular Biology - MOL CELL BIOL. 01/2003; 23(1):131-139.
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Tohru Ishitani,
Jun Ninomiya-Tsuji,
Shin-ichi Nagai,
Michiru Nishita,
Marc Meneghini,
Nick Barker,
Marian Waterman,
Bruce Bowerman,
Hans Clevers,
Hiroshi Shibuya,
Kunihiro Matsumoto
Nature 06/1999; 399(6738):798-802. · 36.28 Impact Factor
