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Nobuyuki Itoh

Publications and source records attributed to Nobuyuki Itoh.

At least 19 recordsLinked to original sources
PubMed2006

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish.

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.

Amino Acid Sequence↗
PubMed2006

Fgf16 is essential for pectoral fin bud formation in zebrafish.

Zebrafish pectoral fin bud formation is an excellent model for studying morphogenesis. Fibroblast growth factors (Fgfs) and sonic hedgehog (shh) are essential for pectoral fin bud formation. We found that Fgf16 was expressed in the apical ectodermal ridge (AER) of fin buds. A knockdown of Fgf16 function resulted in no fin bud outgrowth. Fgf16 is required for cell proliferation and differentiation in the mesenchyme and the AER of the fin buds, respectively. Fgf16 functions downstream of Fgf10, a mesenchymal factor, signaling to induce the expression of Fgf4 and Fgf8 in the AER. Fgf16 in the AER and shh in the zone of polarizing activity (ZPA) interact to induce and/or maintain each other's expression. These findings have revealed that Fgf16, a newly identified AER factor, plays a crucial role in pectoral fin bud outgrowth by mediating the interactions of AER-mesenchyme and AER-ZPA.

Animals↗
PubMed2006

Fgf21 is essential for haematopoiesis in zebrafish.

Fibroblast growth factors (Fgfs) function as key secreted signalling molecules in many developmental events. The zebrafish is a powerful model system for the investigation of embryonic vertebrate haematopoiesis. Although the effects of Fgf signalling on haematopoiesis in vitro have been reported, the functions of Fgf signalling in haematopoiesis in vivo remain to be explained. We identified Fgf21 in zebrafish embryos. Fgf21-knockdown zebrafish embryos lacked erythroid and myeloid cells but not blood vessels and lymphoid cells. The knockdown embryos had haemangioblasts and haematopoietic stem cells. However, the knockdown embryos had significantly fewer myeloid and erythroid progenitor cells. In contrast, Fgf21 had no significant effect on cell proliferation and apoptosis in the intermediate cell mass. These results indicate that Fgf21 is a newly identified factor essential for the determination of myelo-erythroid progenitor cell fate in vivo.

Animals↗
PubMed2006

Role of Fgf10 in cell proliferation in white adipose tissue.

The development of white adipose tissue (WAT) involves adipogenesis and cell proliferation. Although the adipogenesis has been well studied, the cell proliferation has not. Therefore, we examined the mechanism of the proliferation by analyzing Fgf10(-/-) mouse embryonic WAT, in which adipogenesis and proliferation were severely impaired. D-type cyclin expression and retinoblastoma family protein phosphorylation essential for cell proliferation were examined in WAT. Both cyclin D2 expression and p130 phosphorylation were impaired in the Fgf10(-/-) WAT. In mouse embryonic fibroblasts, Fgf10 stimulated cyclin D2 expression and p130 phosphorylation, which were inhibited by an inhibitor of the Ras/MAPK pathway. These results suggest that Fgf10 stimulates cell proliferation in WAT through the Ras/MAPK pathway followed by the cyclin D2-dependent phosphorylation of p130. In contrast, expression but not phosphorylation of pRb was impaired in the Fgf10(-/-) WAT. As pRb is essential for adipogenesis, Fgf10 might play a role in adipogenesis by inducing its expression.

Adipogenesis↗
PubMed2006

Neudesin, a secreted factor, promotes neural cell proliferation and neuronal differentiation in mouse neural precursor cells.

Neudesin encodes a secreted signal with neurotrophic activity in neurons. Most neurotrophic factors are involved in neural cell proliferation and/or differentiation. However, the role of neudesin in neural development remains to be elucidated. We examined the expression of neudesin in mouse embryonic cerebral cortex and cultured mouse neural precursor cells and its roles in neural development. Neudesin was expressed in the embryonic cerebral cortex early in development. Its expression was observed mainly in the preplate, where mostly postmitotic neural cells existed. Because neudesin mRNA was expressed in the neural precursor cells before the appearance of neurons, the roles of neudesin in neural development were examined by using the precursor cells. Neudesin significantly promoted neuronal differentiation and overrode the undifferentiated state of the neural precursor cells sustained by fibroblast growth factor 2 (FGF2). In contrast, it inhibited the differentiation of astrocytes. In addition, neudesin transiently promoted neural cell proliferation early in the developmental process. The effect on cell proliferation was distinct from that of FGF2, a self-renewal-promoting factor for neural precursor cells. The differentiation was mediated though activation of the protein kinase A (PKA) and phosphatidylinositol-3 kinase (PI-3K) pathways. In contrast, the proliferation was mediated through the mitogen-activated protein kinase and PKA pathways. The expression profile and activity indicate that neudesin plays unique roles in neural development. The present findings have revealed new potential roles of neudesin in neural cell proliferation and neuronal differentiation.

Animals↗
PubMed2005

Fgf19 regulated by Hh signaling is required for zebrafish forebrain development.

Fibroblast growth factor (Fgf) signaling plays important roles in brain development. Fgf3 and Fgf8 are crucial for the formation of the forebrain and hindbrain. Fgf8 is also required for the midbrain to form. Here, we identified zebrafish Fgf19 and examined its roles in brain development by knocking down Fgf19 function. We found that Fgf19 expressed in the forebrain, midbrain and hindbrain was involved in cell proliferation and cell survival during embryonic brain development. Fgf19 was also essential for development of the ventral telencephalon and diencephalon. Regional specification is linked to cell type specification. Fgf19 was also essential for the specification of gamma-aminobutyric acid (GABA)ergic interneurons and oligodendrocytes generated in the ventral telencephalon and diencephalon. The cross talk between Fgf and Hh signaling is critical for brain development. In the forebrain, Fgf19 expression was down-regulated on inhibition of Hh but not of Fgf3/Fgf8, and overexpression of Fgf19 rescued partially the phenotype on inhibition of Hh. The present findings indicate that Fgf19 signaling is crucial for forebrain development by interacting with Hh and provide new insights into the roles of Fgf signaling in brain development.

Amino Acid Sequence↗
PubMed2005

Regulation of mammalian tooth cusp patterning by ectodin.

Mammalian tooth crowns have precise functional requirements but cannot be substantially remodeled after eruption. In developing teeth, epithelial signaling centers, the enamel knots, form at future cusp positions and are the first signs of cusp patterns that distinguish species. We report that ectodin, a secreted bone morphogenetic protein (BMP) inhibitor, is expressed as a "negative" image of mouse enamel knots. Furthermore, we show that ectodin-deficient mice have enlarged enamel knots, highly altered cusp patterns, and extra teeth. Unlike in normal teeth, excess BMP accelerates patterning in ectodin-deficient teeth. We propose that ectodin is critical for robust spatial delineation of enamel knots and cusps.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Neudesin, a novel secreted protein with a unique primary structure and neurotrophic activity.

We identified a novel secreted protein and named it neudesin. Mouse neudesin of 171 amino acids is unique with no primary structural similarity to any known proteins. The neudesin protein produced in cultured cells was secreted efficiently into the culture medium. Mouse neudesin mRNA was expressed abundantly in the developing brain and spinal cord in embryos, but was expressed widely in postnatal tissues including brain, heart, lung, and kidney. Mouse neudesin mRNA was expressed in neurons but not glial cells of the brain. The protein exhibited significant neurotrophic activity in primary cultured mouse neurons but not mitogenic activity in primary cultured mouse astrocytes. Neudesin activated the mitogen-activated protein (MAP) and phosphatidylinositol-3 (PI-3) kinase pathways. The activity of neudesin was inhibited by the inhibitor pertussis toxin for Gi/Go-protein but not by inhibitors for receptor tyrosine kinases. These results indicated that the activity was mediated via the activation of the MAP and PI-3 kinase pathways, potentially by the activation of a Gi/Go-protein-coupled receptor. Human neudesin of 172 amino acids with high similarity ( approximately 91% identity) to mouse neudesin was also identified. The human neudesin gene was mapped to chromosome 1p33. The identification of neudesin, a novel secreted protein with a unique primary structure and neurotrophic activity, will provide new insights into the development and maintenance of neuron

Amino Acid Sequence↗
PubMed2005

Complementary antagonistic actions between C-type natriuretic peptide and the MAPK pathway through FGFR-3 in ATDC5 cells.

We previously reported that C-type natriuretic peptide (CNP) stimulates endochondral ossification and corrects the reduction in body length of achondroplasia model mouse with constitutive active fibroblast growth factor receptor 3 (FGFR-3). In order to examine the interaction between CNP and FGFR-3, we studied intracellular signaling by using ATDC5 cells, a mouse chondrogenic cell line, and found that FGF2 and FGF18 markedly reduced CNP-dependent intracellular cGMP production, and that these effects were attenuated by MAPK inhibitors. Western blot analysis demonstrated that the level of GC-B, a particulate guanylyl cyclase specific for CNP, was not changed by treatment with FGFs. Conversely, CNP and 8-bromo-cGMP strongly and dose-dependently inhibited the induction of ERK phosphorylation by FGF2 and FGF18 without changing the level of FGFR-3, although they did not affect the phosphorylation of STAT-1. In the organ-cultured fetal mouse tibias, CNP and FGF18 counteracted on the longitudinal bone growth, and both the size and number of hypertrophic chondrocytes. The FGF/FGFR-3 pathway is known as the negative regulator of endochondral ossification. We found that FGFs inhibited CNP-stimulated cGMP production by disrupting the signaling pathway through GC-B while CNP antagonized the activation of the MAPK cascade by FGFs. These results suggest that the CNP/GC-B pathway plays an important role in growth plate chondrocytes and constitutes the negative cross talk between FGFs and the activity of MAPK. Our results may explain one of the molecular mechanisms of the growth stimulating action of CNP and suggest that activation of the CNP/GC-B pathway may be effective as a novel therapeutic strategy for achondroplasia.

Animals↗
PubMed2005

Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model.

Parkinson disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic (DA) neurons. ES cells are currently the most promising donor cell source for cell-replacement therapy in PD. We previously described a strong neuralizing activity present on the surface of stromal cells, named stromal cell-derived inducing activity (SDIA). In this study, we generated neurospheres composed of neural progenitors from monkey ES cells, which are capable of producing large numbers of DA neurons. We demonstrated that FGF20, preferentially expressed in the substantia nigra, acts synergistically with FGF2 to increase the number of DA neurons in ES cell-derived neurospheres. We also analyzed the effect of transplantation of DA neurons generated from monkey ES cells into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated (MPTP-treated) monkeys, a primate model for PD. Behavioral studies and functional imaging revealed that the transplanted cells functioned as DA neurons and attenuated MPTP-induced neurological symptoms.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2005

Cloning and expression of a brain-specific putative UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase gene.

We isolated a rat cDNA clone and its human orthologue, which are most homologous to UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase 9, by homology-based PCR from brain. Nucleotide sequence analysis of these putative GalNAc-transferases (designated pt-GalNAc-T) showed that they contained structural features characteristic of the GalNAc-transferase family. It was also found that human pt-GalNAc-T was identical to the gene WBSCR17, which is reported to be in the critical region of patients with Williams-Beuren Syndrome, a neurodevelopmental disorder, and to be predominantly expressed in brain and heart. In order to investigate the expression of pt-GalNAc-T in brain in more detail, we first examined that of human pt-GalNAc-T by Northern blot analysis and found the expression of the 5.0-kb mRNA to be most abundant in cerebral cortex with somewhat less abundant in cellebellum. The expression of rat pt-GalNAc-T was investigated more extensively. The brain-specific expression of 2.0-kb and 5.0-kb transcripts was demonstrated by Northern blot analysis. In situ hybridization in the adult brain revealed high levels of expression in cerebellum, hippocampus, thalamus, and cerebral cortex. Moreover, observation at high magnification revealed the expression to be associated with neurons, but not with glial cells. Analysis of the rat embryos also demonstrated that rat pt-GalNAc-T was expressed in the nervous system, including in the diencephalons, cerebellar primordium, and dorsal root ganglion. However, recombinant human pt-GalNAc-T, which was expressed in insect cells, did not glycosylate several peptides derived from mammalian mucins, suggesting that it may have a strict substrate specificity. The brain-specific expression of pt-GalNAc-T suggested its involvement in brain development, through O-glycosylation of proteins in the neurons.

Amino Acid Sequence↗
PubMed2004

Novel 70-kDa chondroitin sulfate/dermatan sulfate hybrid chains with a unique heterogeneous sulfation pattern from shark skin, which exhibit neuritogenic activity and binding activities for growth factors and neurotrophic factors.

Chondroitin sulfate (CS) and dermatan sulfate (DS) hybrid chains of proteoglycans are critical in growth factor binding, neuritogenesis, and brain development. Here we isolated CS/DS hybrid chains from shark skin aiming to develop therapeutic agents. Digestion with various chondroitinases showed that both GlcUA- and IdoUA-containing disaccharides are scattered along the polysaccharide chains with an unusually large average molecular mass of 70 kDa. The CS/DS chains were separated into major (80%) and minor (20%) fractions by anion-exchange chromatography. Both fractions had relatively low degrees of sulfation (sulfate/disaccharide molar ratio=1.17 versus 0.87), showing a unique feature compared with the marine CS and DS isolated to date, most of which are oversulfated. They were highly heterogeneous and characterized by multiple disaccharides including GlcUA-GalNAc, GlcUA-GalNAc(6S), GlcUA-GalNAc(4S), IdoUA-GalNAc(4S), GlcUA-GalNAc(4S,6S), IdoUA-GalNAc(4S,6S), GlcUA(2S)-GalNAc(6S), and/or IdoUA(2S)-GalNAc(6S), IdoUA(2S)-GalNAc(4S) and novel GlcUA(2S)-GalNAc(4S), where 2S, 4S, and 6S represent 2-O-, 4-O- and 6-O-sulfate, respectively. The CS/DS chains bound two neurotrophic factors and various growth factors expressed in the brain with high affinity as evaluated for the major fraction by kinetic analysis using a surface plasmon resonance detector, and also promoted the outgrowth of neurites of both an axonic and a dendritic nature. The neuritogenic activity was abolished completely by digestion with chondroitinase ABC, AC-I, or B, suggesting the importance of both GlcUA- and IdoUA-containing moieties. It also showed anti-heparin cofactor II activity comparable to that exhibited by DS from porcine skin. Thus, by virtue of its unique structure and biological activities, DS will find a potential use in therapeutics.

Animals↗
PubMed2004

Laminar patterning in the developing neocortex by temporally coordinated fibroblast growth factor signaling.

Laminar organization, a fundamental neural architecture in the CNS, is a prominent feature of the neocortex, where the cortical neurons in spatially distinct layers are generated from the common progenitors in a temporally distinct manner during development. Despite many advances in the characterization of the molecular mechanisms of the radial migration of cortical neurons, the way in which the early-late temporal sequence of cortical neuron generation is linked with the deep-superficial spatial sequence of cell body positioning remains obscure. Using in vivo electroporation-mediated gene transfer, we show here that the activities mediated by fibroblast growth factor receptors (FGFRs) in cortical progenitors are critical for conferring proper migratory properties on nascent neuronal progeny. Furthermore, we provide supportive evidence that Pea3 subfamily members of Ets (Pea3-Ets) transcription factors mediate the activities of FGFR at the mid to late phase of neocortical development. In addition, using FGF18 knock-out mice, we demonstrate that FGF18 expressed by early-generated cortical neurons in the cortical plate is critical for the expression of Pea3-Ets transcription factors and that FGF18 is sufficient to induce their expressions. Our results thus imply that a feedback mechanism mediated by FGF signaling is involved in setting up the proper laminar positioning of cortical neurons; FGF18 derived from early-generated cortical neurons acts on the cortical progenitors expressing FGFRs and induces the expression of Pea3-Ets transcription factors that, in turn, confer proper migratory behaviors on nascent cortical progeny during the mid to late stages of neocortical development.

Animals↗
PubMed2004

Fgf18 is required for embryonic lung alveolar development.

Fgf18 is abundantly expressed in mouse embryonic lungs. To elucidate the roles of Fgf18 in mouse embryonic lung development, we examined the Fgf18-/- embryonic lungs. Although the sizes of the Fgf18-/- lungs were a little smaller in appearance than those of wild-type lungs, neither proximal nor distal airway branching in the Fgf18-/- lungs was impaired. However, the Fgf18-/- lungs at E18.5 had reduced alveolar space, thicker interstitial mesenchymal compartments, and many embedded capillaries. Cell proliferation in the Fgf18-/- lungs was also transiently reduced around E17.5, although the expression of marker genes for lung epithelial cells in the Fgf18-/- lungs was not impaired. The present findings indicate that the Fgf18 plays roles in lung alveolar development during late embryonic lung development stages. The cell proliferation during the terminal saccular stage stimulated by Fgf18 might play roles in the remodeling of the distal lung.

Animals↗
PubMed2004

Structural and functional characterization of oversulfated chondroitin sulfate/dermatan sulfate hybrid chains from the notochord of hagfish. Neuritogenic and binding activities for growth factors and neurotrophic factors.

Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of D-glucuronic acid (GlcUA)- and L-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite outgrowth-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic factors (viz. fibroblast growth factor-2, -10, -16, and -18; midkine; pleiotrophin; heparin-binding epidermal growth factor-like growth factor; vascular endothelial growth factor; brain-derived neurotrophic factor; and glial cell line-derived neurotrophic factor), most of which are expressed in the brain, although fibroblast growth factor-1 and ciliary neurotrophic factor showed no binding. Kinetic analysis revealed high affinity binding of these growth factors and, for the first time, of the neurotrophic factors. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these growth factors, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these growth factors. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765-9776), suggest physiological roles of the hybrid chains in the development of the brain.

Animals↗
PubMed2004

Biochemical analysis of pathogenic ligand-dependent FGFR2 mutations suggests distinct pathophysiological mechanisms for craniofacial and limb abnormalities.

Gain-of-function missense mutations in FGF receptor 2 (FGFR2) are responsible for a variety of craniosynostosis syndromes including Apert syndrome (AS), Pfeiffer syndrome (PS) and Crouzon syndrome (CS). Unlike the majority of FGFR2 mutations, S252W and P253R AS mutations and a D321A PS mutation retain ligand-dependency and are also associated with severe limb pathology. In addition, a recently identified ligand-dependent S252L/A315S double mutation in FGFR2 was shown to cause syndactyly in the absence of craniosynostosis. Here, we analyze the effect of the canonical AS mutations, the D321A PS mutation and the S252L/A315S double mutation on FGFR2 ligand binding affinity and specificity using surface plasmon resonance. Both AS mutations and the D321A PS mutation, but not the S252L/A315S double mutation, increase the binding affinity of FGFR2c to multiple FGFs expressed in the cranial suture. Additionally, all four pathogenic mutations also violate FGFR2c ligand binding specificity and enable this receptor to bind FGF10. Based on our data, we propose that an increase in mutant FGFR2c binding to multiple FGFs results in craniosynostosis, whereas binding of mutant FGFR2c to FGF10 results in severe limb pathology. Structural and biophysical analysis shows that AS mutations in FGFR2b also enhance and violate FGFR2b ligand binding affinity and specificity, respectively. We suggest that elevated AS mutant FGFR2b signaling may account for the dermatological manifestations of AS.

Craniofacial Abnormalities↗
PubMed2004

Roles of fibroblast growth factor 10 (Fgf10) in adipogenesis in vivo.

The development of white adipose tissue (WAT) of Fgf10-/- mouse embryos was greatly impaired. Here, we examined the mechanism of Fgf10 action in adipogenesis in vivo. The proliferative activity in the WAT of Fgf10-/- embryos was greatly decreased. We also examined the expression of transcription factors, C/EBPbeta, C/EBPalpha and PPARgamma, that are important for adipogenesis. Although the expression of C/EBPbeta and PPARgamma in the WAT of Fgf10-/- embryos was greatly decreased, the expression of C/EBPalpha was essentially unchanged. Therefore, we examined their expression in the WAT of C/EBPalpha-/- embryos. Although the expression of C/EBPbeta and PPARgamma in the WAT was greatly decreased, the expression of Fgf10 was essentially unchanged. As these results in vivo appeared to be contradictory to a transcriptional cascade model in vitro that C/EBPbeta induces the expression of PPARgamma and C/EBPalpha reported, we also examined their expression in the WAT of wild type embryos at different developmental stages. The expression of Fgf10 and C/EBPalpha was followed by that of C/EBPbeta and PPARgamma. The present findings indicate that Fgf10 but not C/EBPalpha is required for the proliferation of preadipocytes. In contrast, both Fgf10 and C/EBPalpha acting synergistically in separate, parallel pathways are required for the differentiation. Unexpectedly, the transcriptional cascade of adipogenesis in vivo described here is distinct from the cascade in vitro previously reported.

Adipocytes↗