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Publications (21)

  • [Show abstract] [Hide abstract] ABSTRACT: Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamor phosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for β-oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3’, 5-triiodothyronine (T3) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3-induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3-treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis.
    Article · Feb 2013 · Hereditas
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    Kenjiro Katsu · Norifumi Tatsumi · Daisuke Niki · [...] · Yuji Yokouchi
    [Show abstract] [Hide abstract] ABSTRACT: During development of left-right asymmetry in the vertebrate embryo, Nodal plays a central role for determination of left-handedness. Bone morphogenetic protein (BMP) signaling has an important role for regulation of Nodal expression, although there is controversy over whether BMP signaling has a positive or negative effect on Nodal expression in the chick embryo. As BMP is a morphogen, we speculated that different concentrations might induce different responses in the cells of the lateral plate mesoderm (LPM). To test this hypothesis, we analyzed the effects of various concentrations of BMP4 and NOGGIN on Nodal expression in the LPM. We found that the effect on Nodal expression varied in a complex fashion with the concentration of BMP. In agreement with previous reports, we found that a high level of BMP signaling induced Nodal expression in the LPM, whereas a low level inhibited expression. However, a high intermediate level of BMP signaling was found to suppress Nodal expression in the left LPM, whereas a low intermediate level induced Nodal expression in the right LPM. Thus, the high and the low intermediate levels of BMP signaling up-regulated Nodal expression, but the high intermediate and low levels of BMP signaling down-regulated Nodal expression. Next, we sought to identify the mechanisms of this complex regulation of Nodal expression by BMP signaling. At the low intermediate level of BMP signaling, regulation depended on a NODAL positive-feedback loop suggesting the possibility of crosstalk between BMP and NODAL signaling. Overexpression of a constitutively active BMP receptor, a constitutively active ACTIVIN/NODAL receptor and SMAD4 indicated that SMAD1 and SMAD2 competed for binding to SMAD4 in the cells of the LPM. Nodal regulation by the high and low levels of BMP signaling was dependent on Cfc up-regulation or down-regulation, respectively. We propose a model for the variable effects of BMP signaling on Nodal expression in which different levels of BMP signaling regulate Nodal expression by a balance between BMP-pSMAD1/4 signaling and NODAL-pSMAD2/4 signaling.
    Full-text Article · Nov 2012 · Developmental Biology
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    Kenjiro Katsu · Daisuke Tokumori · Norifumi Tatsumi · [...] · Yuji Yokouchi
    [Show abstract] [Hide abstract] ABSTRACT: During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.
    Full-text Article · Dec 2011 · Developmental Biology
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    Dataset: Figure S1
    [Show abstract] [Hide abstract] ABSTRACT: Expression of Cerberus/Dan family and the construct used. A, B) mCerberus 1 homolog (Cer1), Dan and Prdc genes are expressed in the ureteric bud (U) and kidney mesenchyme (KM) of E11.5 embryos and whole embryonic kidneys (K) at E12.5 and E15.5, as revealed by RT-PCR. Note that Cer1 expression is elevated in the ureteric bud in the transgenic embryonic kidney (Cer1, star) relative to the wild-type (Wt) at E11.5 (compare B to A). Like Cer1, Prdc expression is elevated due to the gain of function of Cer1 expression. The DAN/Cerberus genes, Dan and Prdc are also expressed in the developing kidney. C, D) Whole mount in situ hybridization shows that Cer1 is expressed in both the ureteric bud and kidney mesenchyme at E11.75 and E12.5. E. Schematic structure of the construct used to express Cer1 and eGFP in the ureteric bud. F-H) Wild-type kidneys prepared from embryos at the E11.5, E15.5 and newborn (NB) stages. I-K) Pax2 promoter-driven GFP can be detected in the ureteric bud. The arrow in (I) indicates the ureteric bud of the E11.5 embryonic kidney. NB; newborn. Scale bar, 100 µm. (TIF)
    Full-text Dataset · Nov 2011
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    Dataset: Table S2
    [Show abstract] [Hide abstract] ABSTRACT: Primers used to analyse changes in gene expression induced by Cer1+. (DOC)
    Full-text Dataset · Nov 2011
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    Dataset: Movie S2
    [Show abstract] [Hide abstract] ABSTRACT: Visualization of Cer1-induced changes in development of the ureteric bud tree during kidney organogenesis analysed. The ureteric bud was identified with antibodies against cytokeratin at E15.5 by using optical projection tomography (OPT). A kidney of a wild-type (Wt) embryo on the left and the one expressing Cer1 in the ureteric bud on the right side identifies Cer1 induced changes in the structure of the ureteric tree. (MOV)
    Full-text Dataset · Nov 2011
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    Dataset: Movie S1
    [Show abstract] [Hide abstract] ABSTRACT: Cer1 expression changes the overall pattern of ureteric bud branching when compared to controls. The kidney primordial were prepared at E11.5 and subjected to organ culture for 120 hrs. Ureteric was visualized by genetic means by yellow fluorescent protein that was activated from the floxed Rosa26 locus as a result of HoxB7Cre recombination. Analysis of the time-lapse recordings reveal that Cer1 gain of function in the ureteric bud shifts the mode of ureteric bud branching from a difurgation type towards the trifurgation one (see also Figure S4). C, D) Later Cer1 over expressing kidneys have a tendency to develop also lateral side branches not that typical in the control. As a result the overall pattern formation of the ureteric bud of the kidneys that were prepared from the Cer1 kidneys appears different from the control. (MOV)
    Full-text Dataset · Nov 2011
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    Dataset: Figure S4
    [Show abstract] [Hide abstract] ABSTRACT: Still images from time-lapse movies from YFP+ ureteric buds of wild-type and Cer1+ embryonic kidneys. The still images from cultures of E115 kidneys were used to evaluate the influence of Cer1 on the mode of bi/trifurcation and generation of the ureteric bud branches. Note that Cer1 gain of functions has promoted ureteric bud development already at 00 hr (compare B to A, arrowhead), trifurcation of the bud at 24 hr time point (compare D to C, arrowheads) and changes in the overall mode of ureteric branching when compared to the pattern of the ureteric three in later stage cultures of HoxB7Cre;RYFPR26 marked ureteric bud (compare the ureteric tree pattern in F,H,J with E,G,I). (TIF)
    Full-text Dataset · Nov 2011
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    Dataset: Figure S3
    [Show abstract] [Hide abstract] ABSTRACT: Cer1 has a positive effect on the length of the early ureteric bud branches. The kidneys were prepared at E11.5 from embryos that had either YFP only or both the YFP and Cer1 genes (see the methods for details). The length of each ureteric bud branch during early stages of kidney development was calculated according to Watanabe and Costantini (2004) [83] analyzed from still images made from the time-lapse movies recorded of the cultured kidneys. Cer1 has stimulated to a certain degree the length of early branches. At 48 hrs of culture the branches from the 2nd to 5th generations appear measurable longer. (TIF)
    Full-text Dataset · Nov 2011
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    [Show abstract] [Hide abstract] ABSTRACT: The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.
    Full-text Article · Nov 2011 · PLoS ONE
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    Dataset: Figure S5
    [Show abstract] [Hide abstract] ABSTRACT: Cer1+ has changed distance of first ureteric bud branch points from the mean centre. The embryonic kidneys were prepared at E15.5 from wild-type or Cer1 embryos, stained as whole mounts with anti-cytokertin antibody and subjected to analysis of the three dimensional structure of the ureteric tree with optical projection tomography. The morphometric analysis reveal that Cer1 expression diminishes n several samples the distance of the first ureteric bud branch points from the mean center or the kidney when compared to the same parameter values the wild-type (Wt) kidney. (TIF)
    Full-text Dataset · Nov 2011
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    Dataset: Table S1
    [Show abstract] [Hide abstract] ABSTRACT: Primers used to genotype the generated Cer1+ mouse lines. (DOC)
    Full-text Dataset · Nov 2011
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    Dataset: Figure S2
    [Show abstract] [Hide abstract] ABSTRACT: Cer1 gain of function has enlarged the kidney. A) The kidney of a five-month-old wild-type mouse. B) A kidney that has expressed Cer1 in the ureteric bud. C, D) Sections from the kidneys shown in (A, B). Counting the volume of the kidney in six similar samples shown in (A and B) indicates that the kidney that had expressed Cer1 is larger in size than the wild-type control kidney (Wt) (E). Bar 500 µm. (TIF)
    Full-text Dataset · Nov 2011
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    Dataset: Figure S7
    [Show abstract] [Hide abstract] ABSTRACT: Schematic representation of the potential mode of action of Cer1 in the control of ureteric branching. As a secreted protein, the Cer1 protein binds Bmp2/4 proteins in the ureteric bud and the kidney mesenchyme but not Gdnf. Bmp2 and Bmp4 have both been implicated as inhibitory signals for ureteric bud branching involving Alk3 receptor in the ureteric bud. Bmp4 signaling normally leads to repression of the expression of Gdnf, which signals via its Ret receptor expressed in the ureteric bud and promotes ureteric bud development via positive feedback signaling with Wnt11. Lower activity of Bmp due to Cer1 mediated inhibition enhances Gdnf expression and this promotes ureteric bud branching by stimulation of the positive signaling loop between Gdnf and Wnt11 promoting ureteric bud development. Cer1 inhibited to a moderate level canonical β-catenin mediated Wnt signaling and this may be relevant in advancing initiation of branching at the tip region. Modulation of Bmp by Cer1 may also influence kidney mesenchyme which seen changes Wnt4 expression controlling nephrogenesis. Depending of the level of Bmp4 and Cer1, Bmp4 either inhibits or induces Wnt4 expression. (TIF)
    Full-text Dataset · Nov 2011
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    Dataset: Figure S6
    [Show abstract] [Hide abstract] ABSTRACT: Cer1 loss and gain of function influences Bmp expression and signaling. Real-time PCR analysis of total RNA isolated from kidneys of Cer1 heterozygous (+/-knockout (-/-) newborn mice demonstrate reduced expression of Bmp4 (A), Bmp2 (B) and Wnt4 (C) gene expression. D) The pSmad protein revealed by a specific antibody indicates activity in developing nephrons and ureteric bud and reduction in these sites due to Cer1 gain of function (compare E with D). F) pSmad remains expressed in the cortex of the kidney in case of Cer1 knock out (-/-). D-F, Bar 10 µm. (TIF)
    Full-text Dataset · Nov 2011
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    Daisuke Niki · Kenjiro Katsu · Yuji Yokouchi
    [Show abstract] [Hide abstract] ABSTRACT: Angiopoietin-like proteins (ANGPTLs) are secreted proteins possessing an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen-like domain and are known as angiogenic factors. Several members of ANGPTLs also regulate lipid metabolism independently of angiogenic effects, but most of their functions during vertebrate development are not demonstrated. To ascertain their developmental functions, we examined the expression patterns of Angptl1, 2, 3, 4, 5, and 7 orthologues during chick development using whole-mount in situ hybridization. Angptl1 was first detected at embryonic day 3 (E3) in the somite. At E4, Angptl1 was expressed in somite-derivatives and limb mesenchyme. Angptl2 was first detected at E3 in the hindbrain. At E4, Angptl2 was expressed in neuroepithelium of forebrain and hindbrain and partly in the heart. Angptl3 was first detected at E3 and continued to be expressed in the liver and yolk sac at E4. Angptl4 was first detected at E3 in the somites and liver. At E4, Angptl4 was also observed in the heart. Angptl5 was not detected in these developmental stages. Angptl7 was first detected at E3 in the ectoderm overlying the lenses of the eyes. At E4, Angptl7 was specifically expressed in cornea. These data suggest that each member of the ANGPTL family could be related to angiogenesis during various organogeneses of the developing chick embryo.
    Full-text Article · Dec 2009 · Development Growth and Regeneration
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    [Show abstract] [Hide abstract] ABSTRACT: Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.
    Full-text Article · May 2008 · Developmental Dynamics
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    Norifumi Tatsumi · Rika Miki · Kenjiro Katsu · Yuji Yokouchi
    [Show abstract] [Hide abstract] ABSTRACT: During chick liver development, the liver bud arises from the foregut, invaginates into the septum transversum, and elongates along and envelops the ductus venosus. However, the mechanism of liver bud migration is only poorly understood. Here, we demonstrate that a GDNF family ligand involved in neuronal outgrowth and migration, neurturin (NRTN), and its receptor, GFRalpha2, are essential for liver bud migration. In the chick embryo, we found that GFRalpha2 was expressed in the liver bud and that NRTN was expressed in the endothelial cells of the ductus venosus. Inhibition of GFRalpha2 signaling suppressed liver bud elongation along the ductus venous without affecting cell proliferation and apoptosis. Moreover, ectopic expression of NRTN perturbed the directional migration along the ductus venosus, leading to splitting or ectopic branching of the liver. We showed that liver buds selectively migrated toward an NRTN-soaked bead in vitro. These data represent a new model for liver bud migration: NRTN secreted from endothelial cells functions as a chemoattractant to direct the migration of the GFRalpha2-expressing liver bud in early liver development.
    Full-text Article · Aug 2007 · Developmental Biology
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    Takahiro Yamanishi · Kenjiro Katsu · Jun-Ichi Funahashi · [...] · Yuji Yokouchi
    [Show abstract] [Hide abstract] ABSTRACT: During vertebrate inner ear development, compartmentalization of the auditory and vestibular apparatuses along two axes depends on the patterning of transcription factors expressed in a region-specific manner. Although most of the patterning is regulated by extrinsic signals, it is not known how Nkx5.1 and Msx1 are patterned. We focus on Dan, the founding member of the Cerberus/Dan gene family that encodes BMP antagonists, and describe its function in morphogenesis and patterning. First, we confirmed that Dan is expressed in the dorso-medial region of the otic vesicle that corresponds to the presumptive endolymphatic duct and sac (ed/es). Second, we used siRNA knockdown to demonstrate that depletion of Dan induced both a severe reduction in the size of the ed/es and moderate deformities of the semicircular canals and cochlear duct. Depletion of Dan also caused suppression of Nkx5.1 in the dorso-lateral region, suppression of Msx1 in the dorso-medial region, and ectopic induction of Nkx5.1 and Msx1 in the ventro-medial region. Most of these phenotypes also appeared following misexpression of the constitutively active form of BMP receptor type Ib. Thus, Dan is required for the normal morphogenesis of the inner ear and, by inhibiting BMP signaling, for the patterning of the transcription factors Nkx5.1 and Msx1.
    Full-text Article · Feb 2007 · Development Growth and Regeneration
  • K. Katsu · D. Tokumori · N. Tatsumi · [...] · Y. Yokouchi
    Article · Sep 2005 · Mechanisms of Development