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Kazuki Nakao

Publications and source records attributed to Kazuki Nakao.

At least 19 recordsLinked to original sources
PubMed2006

DNA methylation regulates long-range gene silencing of an X-linked homeobox gene cluster in a lineage-specific manner.

DNA methylation is a major epigenetic mechanism that has been suggested to control developmental gene regulation during embryogenesis, but its regulatory mechanisms remain unclear. In this report, we show that CpG islands associated with the X-linked homeobox gene cluster Rhox, which is highly expressed in the extraembryonic trophectoderm, are differentially methylated in a stage- and lineage-specific manner during the post-implantation development of mice. Inactivation of both Dnmt3a and Dnmt3b, DNA methyltransferases essential for the initiation of de novo DNA methylation, abolished the establishment of DNA methylation and the silencing of Rhox cluster genes in the embryo proper. The Dnmt3-dependent CpG-island methylation at the Rhox locus extended for a large genomic region ( approximately 1 Mb) containing the Rhox cluster and surrounding genes. Complementation experiments using embryonic stem (ES) cells deficient in the DNA methyltransferases suggested that the CpG-island methylation by Dnmt3a and Dnmt3b was restricted within this large genomic region, and did not affect the neighboring genes outside it, implicating the existence of region-specific boundaries. Our results suggest that DNA methylation plays important roles in both long-range gene silencing and lineage-specific silencing in embryogenesis.

Animals↗
PubMed2006

Essential pro-Bmp roles of crossveinless 2 in mouse organogenesis.

We here report essential roles of the Bmp-binding protein crossveinless 2 (Cv2; Bmper) in mouse organogenesis. In the null Cv2 mutant mouse, gastrulation occurs normally, but a number of defects are found in Cv2-expressing tissues such as the skeleton. Cartilage differentiation by Bmp4 treatment is reduced in cultured Cv2(-/-) fibroblasts. Moreover, the defects in the vertebral column and eyes of the Cv2(-/-) mouse are substantially enhanced by deleting one copy of the Bmp4 gene, suggesting a pro-Bmp role of Cv2 in the development of these organs. In addition, the Cv2(-/-) mutant exhibits substantial defects in Bmp-dependent processes of internal organ formation, such as nephron generation in the kidney. This kidney hypoplasia is synergistically enhanced by the additional deletion of Kcp (Crim2) which encodes a pro-Bmp protein structurally related to Cv2. This study demonstrates essential pro-Bmp functions of Cv2 for locally restricted signal enhancement in multiple aspects of mammalian organogenesis.

Animals↗
PubMed2006

B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency.

The ontogenic source of definitive hematopoietic system has been identified in non-mammalian vertebrates such as birds and amphibians by orthotopic embryo grafting, but remains unclear for mammals because of technical difficulties. Here, we successfully generated mouse chimeras by grafting yolk sac (YS) on YS of the host embryos before establishing circulation between YS and embryo proper and cultured the whole embryo for 66 h. Donor YS were isolated from C57BL/6 Ly-5.1 and EGFP-transgenic mouse embryos, and recipient embryos from C57BL/6 Ly-5.2 mouse. Almost one-half of the grafts in YS-YS chimeras survived and had obvious blood flow; graft-derived cells comprised 12.7+/-0.9% of the blood cells in the circulation. These graft-derived blood cells consisted mainly of erythroid cells, some myeloid cells and a few blastic cells. In addition, CD19(+) B cells were generated from the graft-derived cells isolated from aorta-gonad-mesonephros (AGM) regions of the YS-YS chimeras; however, the frequency of the YS-derived B cell was low (1.0+/-0.6%) when co-cultured with OP9 stromal cells. These results demonstrate that B cell potential exists in YS before the circulation. Although the major source for B cell is intra-embryonic AGM region, YS may contribute to definitive lymphopoiesis in vivo in mice.

Animals↗
PubMed2006

Neutrophil direction sensing and superoxide production linked by the GTPase-activating protein GIT2.

In neutrophils, superoxide anion production generally accompanies chemotaxis and functions in killing invading pathogens. The GIT2 GTPase-activating protein binds to the guanine nucleotide-exchange factor alphaPIX. Here we show that GIT2 was necessary for directional chemotaxis and for the suppression of superoxide production in G protein-coupled receptor-stimulated neutrophils. GIT2 was also necessary for the orientation of superoxide production toward chemoattractant sources. GIT2 suppressed the activity of ADP ribosylation factor 1 and was a component of the Gbetagamma subunit-mediated direction-sensing machinery 'downstream' of G protein-coupled receptor signaling. This study establishes a function for GIT2 in linking chemotaxis and superoxide production in neutrophils and shows that loss of GIT2 in vivo leads to an immunodeficient state.

ADP-Ribosylation Factor 1↗
PubMed2005

Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture.

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+ E-cadherin(ECD)+ PDGFRalpha(alphaR)+ population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ ECD+ alphaR- and Gsc+ ECD- alphaR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages, respectively. The presence of mesendodermal cells in nascent Gsc+ ECD+ alphaR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.

Activins↗
PubMed2005

Farnesylation of retinal transducin underlies its translocation during light adaptation.

G proteins are posttranslationally modified by isoprenylation: either farnesylation or geranylgeranylation. The gamma subunit of retinal transducin (Talpha/Tbetagamma) is selectively farnesylated, and the farnesylation is required for light signaling mediated by transducin in rod cells. However, whether and how this selective isoprenylation regulates cellular functions remain poorly understood. Here we report that knockin mice expressing geranylgeranylated Tgamma showed normal rod responses to dim flashes under dark-adapted conditions but exhibited impaired properties in light adaptation. Of note, geranylgeranylation of Tgamma suppressed light-induced transition of Tbetagamma from membrane to cytosol, and also attenuated its light-dependent translocation from the outer segment to the inner region, an event contributing to retinal light adaptation. These results indicate that, while the farnesylation of transducin is interchangeable with the geranylgeranylation in terms of the light signaling, the selective farnesylation is important for visual sensitivity regulation by providing sufficient but not excessive membrane anchoring of Tbetagamma.

Adaptation, Ocular↗
PubMed2005

Deficiency of protein kinase Calpha in mice results in impairment of epidermal hyperplasia and enhancement of tumor formation in two-stage skin carcinogenesis.

We generated a mouse strain lacking protein kinase Calpha (PKCalpha) and evaluated the significance of the enzyme in epithelial hyperplasia and tumor formation. PKCalpha-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,12-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. Tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKCalpha suppresses tumor promotion. However, the severity of epidermal hyperplasia induced by topical TPA treatment was markedly reduced. In mutant mice, the number of 5-bromo-2'-deoxyuridine-labeled epidermal basal keratinocytes increased 16 to 24 hours after topical TPA treatment as in the case of wild-type mice, but significantly decreased at 36 and 48 hours. Furthermore, the regenerating epithelium induced by skin wound significantly decreased in thickness but was not structurally impaired. The enhanced tumor formation may not be associated with epidermal hyperplasia. The induction levels of epidermal growth factor (EGF) receptor ligands, tumor growth factor alpha (TGF-alpha), and heparin-binding EGF-like growth factor, in the skin of mutant mice by TPA treatment were significantly lower than those in the skin of wild-type mice. PKCalpha may regulate the supply of these EGF receptor ligands in basal keratinocytes, resulting in a reduced epidermal hyperplasia severity in the mutant mice. We propose that PKCalpha positively regulates epidermal hyperplasia but negatively regulates tumor formation in two-stage skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2005

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm.

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.

Animals↗
PubMed2005

A gene-targeted mouse model for chorea-acanthocytosis.

Chorea-acanthocytosis (CHAC) is a hereditary neurodegenerative disorder with autosomal recessive transmission, in which selective degeneration of striatum has been reported in brain pathology. Clinically, CHAC shows Huntington's disease-like neuropsychiatric symptoms and red blood cell acanthocytosis. Recently, we identified the gene, CHAC, encoding a novel protein, chorein, in which a deletion mutation was found in Japanese families with CHAC. In the present study, we have identified the mouse CHAC cDNA sequence and the exon-intron structures of the gene and produced a CHAC model mouse introducing no. 60-61 exon deletion corresponding to a human disease mutation by a gene-targeting technique. The mice began to show acanthocytosis and motor disturbance in old age. In behavioral observations, locomotor activity was significantly decreased and the contact time at social interaction test was decreased significantly in the model mice. In the brain pathology, many apoptotic cells were observed in the striatum of the mutant mice. In neurochemical determinations, the dopamine metabolite, homovanillic acid, concentration decreased significantly in the portion including the midbrain of the mutant mice. These findings are consistent with the human results reported elsewhere and indicate that the CHAC model mice showed a mild phenotype with late adult onset. The CHAC model mouse therefore provides a good model system to study the human disease.

Acanthocytes↗
PubMed2005

Membrane fixation of vascular endothelial growth factor receptor 1 ligand-binding domain is important for vasculogenesis and angiogenesis in mice.

Vascular endothelial growth factor (VEGF) regulates vasculogenesis and angiogenesis by using two tyrosine kinase receptors, VEGFR1 and VEGFR2. VEGFR1 null mutant mice die on embryonic day 8.5 (E8.5) to E9.0 due to an overgrowth of endothelial cells and vascular disorganization, suggesting that VEGFR1 plays a negative role in angiogenesis. We previously showed that the tyrosine kinase (TK) domain of VEGFR1 is dispensable for embryogenesis, since VEGFR1 TK-deficient mice survived and were basically healthy. However, the molecular basis for this is not yet clearly understood. To test the hypothesis that the specific role of VEGFR1 during early embryogenesis is to recruit its ligand to the cell membrane, we deleted the transmembrane (TM) domain in TK-deficient VEGFR1 mice. Surprisingly, about half of the VEGFR1(TM-TK)-deficient mice succumbed to embryonic lethality due to a poor development of blood vessels, whereas other mice were healthy. In VEGFR1(TM-TK)(-/-) mice with growth arrest, membrane-targeted VEGF was reduced, resulting in the suppression of VEGFR2 phosphorylation. Furthermore, the embryonic lethality in VEGFR1(TM-TK)(-/-) mice was significantly increased to 80 to 90% when the genotype of VEGFR2 was changed from homozygous (+/+) to heterozygous (+/-) in 129/C57BL6 mice. These results strongly suggest that the membrane-fixed ligand-binding region of VEGFR1 traps VEGF for the appropriate regulation of VEGF signaling in vascular endothelial cells during early embryogenesis.

Animals↗
PubMed2005

Vascular endothelial growth factor A (VEGF-A) is involved in guidance of VEGF receptor-positive cells to the anterior portion of early embryos.

The hemangioblast in the mesoderm gives rise to both angioblasts and hematopoietic stem cells. The movement of hemangioblast precursor cells in the fetal trunk is a critical event in early embryogenesis. Vascular endothelial growth factor (VEGF) signaling is likely involved in this migration given the partial disturbance of VEGF receptor (VEGFR)-positive cell accumulation and migration in VEGFR2 null mice or mice with a truncated VEGFR1. However, it is not clear how the VEGF system regulates this migration or its direction. We show here that the expression of VEGF-A is dominant in the anterior portion of the embryo, whereas VEGFR1 and VEGFR2 are expressed in the posterior portion of the embryo. An inhibitor of VEGFR kinase blocked the migration of VEGFR-positive cells in a whole-embryo culture system. In addition, VEGFR-positive cells migrated toward a VEGFR1- or VEGFR2-specific ligand in vitro. Furthermore, VEGFR-positive cells derived from wild-type or VEGFR2(+/-) mice moved rapidly anteriorly, whereas cells derived from VEGFR2(+/-) mice carrying a truncated VEGFR1 [VEGFR1(TM-TK)(-/-)] migrated little when injected into wild-type mice. These results suggest that the VEGF-A protein concentrated in the anterior region plays an important role in the guidance of VEGFR-positive cells from the posterior portion to the head region by interacting with VEGFR in the mouse embryo.

Animals↗
PubMed2005

Gene disruption/knock-in analysis of mONT3: vector construction by employing both in vivo and in vitro recombinations.

We report the isolation, spatial/temporal expression and gene disruption phenotype of the mouse ONT3 (mONT3) gene, which encodes a novel secreted signaling protein belonging to the Olfactomedin/Noelin/Tiarin family. During early embryogenesis, mONT3 is detected in the proximal region of the allantois on embryonic day (E) 7.25, in the lateral plate mesoderm on E 8.0 and in the CNS and heart on E 8.5. The homozygous mutant is born normal and fertile. For the expression pattern and loss-of-function analyses, we have successfully generated the LacZ-knock-in targeting vector directly from BACs carrying mouse genomic fragments by combining in vivo and in vitro recombination techniques. This approach enables rapid and reproducible construction of the fully functional vectors within two weeks without the use of restriction enzyme digestion and ligation, or the use of PCR-amplification of large genomic fragments. In addition, this method is applicable to rapid generation of transgenic vectors, demonstrating its versatility in reverse genetic studies.

Alleles↗
PubMed2004

Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons.

fez-like (fezl) is a forebrain-expressed zinc finger gene required for the formation of the hypothalamic dopaminergic and serotonergic (monoaminergic) neurons in zebrafish. To reveal its function in mammals, we analyzed the expression of the mouse orthologue of fezl and generated fezl-deficient mice by homologous recombination. Mouse fezl was expressed specifically in the forebrain from embryonic day 8.5. At mid-gestation, fezl expression was detected in subdomains of the forebrain, including the dorsal telencephalon and ventral diencephalon. Unlike the zebrafish fezl mutant too few, the fezl-deficient mice displayed normal development of hypothalamic monoaminergic neurons, but showed abnormal "hyperactive" behavior. In fezl(-/-) mice, the thalamocortical axons (TCA) were reduced in number and aberrantly projected to the cortex. These mutants had a reduced number of subplate neurons, which are involved in guiding the TCA from the dorsal thalamus, although the subplate neurons were born normally. These results suggest that fezl is required for differentiation or survival of the subplate neurons, and reduction of the subplate neurons in fezl-deficient mice leads to abnormal development of the TCA, providing a possible link between the transcriptional regulation of forebrain development and hyperactive behavior.

Animals↗
PubMed2004

Altered agonist sensitivity and desensitization of neuronal mGluR1 responses in knock-in mice by a single amino acid substitution at the PKC phosphorylation site.

mGluR1 and mGluR5 of the metabotropic glutamate receptor family are coupled to inositol trisphosphate-Ca2+ signal cascades and evoke distinct Ca2+ responses in neural cells and heterologously expressing cells. In heterologous cells, stimulation of recombinant mGluR1 evokes a single-peaked Ca2+ response whereas mGluR5 elicits an oscillatory Ca2+ response. The distinct Ca2+ responses are interchangeable by single amino substitution of aspartate for threonine at the corresponding position of the carboxy-terminal cytoplasmic regions of mGluR1 and mGluR5, respectively. In this investigation, we generated knock-in mice, termed mGluR1 D854T mice, in which aspartate of mGluR1 was replaced with threonine. We examined the effect of this D854T substitution on Ca2+ and current responses mediated by mGluR1 in cultured cerebellar Purkinje cells. Stimulation of mGluR1 D854T by a group 1 mGluR agonist, 3,5-dihydroxyphenylglycine (DHPG) evoked, as in wild-type mGluR1, only single-peaked Ca2+ responses as measured by Ca2+ fluorometric analysis. We then examined mGluR1-induced inward currents carried by nonselective cation channels during whole-cell recordings from cultured Purkinje cells. The mGluR1 D854T mutation abolished the responsiveness of mGluR1 to low concentrations of DHPG (0.5-500 nM) and reduced its desensitization during prolonged agonist application. mGluR1 D854T homozygous mutants showed no apparent behavioural abnormality as analysed by motor movement tests. These results indicate that, although additional modulatory mechanisms seem to be required to produce oscillatory Ca2+ responses of mGluR1, the single amino acid substitution at position 854 of mGluR1 is capable of influencing the kinetics of neuronal mGluR1 responses, most probably through PKC-mediated phosphorylation.

Amino Acid Sequence↗
PubMed2003

Disruption of protein kinase Ceta results in impairment of wound healing and enhancement of tumor formation in mouse skin carcinogenesis.

We have generated a mouse strain lacking protein kinase C (PKC) eta to evaluate its significance in epithelial organization and tumor formation. The PKCeta-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,12-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. The tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKCeta suppresses tumor promotion. Epidermal hyperplasia induced by topical TPA treatment was prolonged in the mutant mice. The enhanced tumor formation may be closely associated with the prolonged hyperplasia induced by topical TPA treatment. In the mutant mice, after inflicting injury by punch biopsy, wound healing on the dorsal skin, particularly reepithelialization, was significantly delayed and impaired in structure. Impairment of epithelial regeneration in wound healing indicates a possibility that PKCeta plays a role in maintenance of epithelial architecture. Homeostasis in epithelial tissues mediated by PKCeta is important for tumor formation in vivo. We propose that PKCeta is involved in tumor formation modulated by regulation of proliferation and remodeling of epithelial cells in vivo.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2003

Impaired lymphopoiesis and altered B cell subpopulations in mice overexpressing Lnk adaptor protein.

Lnk is an adaptor protein expressed primarily in lymphocytes and hemopoietic precursor cells. Marked expansion of B lineage cells occurs in lnk(-/-) mice, indicating that Lnk regulates B cell production by negatively controlling pro-B cell expansion. In addition, lnk(-/-) hemopoietic precursors have an advantage in repopulating the hemopoietic system of irradiated host animals. In this study, we show that Lnk overexpression results in impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. The representation of both B lineage and T lineage cells was reduced in transgenic mice overexpressing Lnk under the control of a lymphocyte-specific expression vector. Whereas the overall number of B and T cells was correlated with Lnk protein expression levels, marginal zone B cells in spleen and B1 cells in the peritoneal cavity were relatively resistant to Lnk overexpression. The C-terminal tyrosine residue, conserved among Lnk family adaptor proteins, was dispensable for the negative regulatory roles of Lnk in lymphocyte development. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Pioglitazone improves the phenotype and molecular defects of a targeted Pkd1 mutant.

Mutations of either PKD1 or PKD2 are associated with autosomal dominant polycystic kidney disease (ADPKD). The molecular function of the gene product of PKD1, polycystin-1, in vitro has been elucidated recently, but the molecular pathological consequences of the loss of polycystin-1 in vivo have remained unclear. We have generated a mouse with a targeted deletion of exons 2-6 of Pkd1 to study the molecular defects in Pkd1 mutants. Homozygote embryos (Pkd1(-/-)) developed hydrops, cardiac conotruncal defects and renal cystogenesis. Total protein levels of beta-catenin in heart and kidney and c-MYC in heart were decreased in Pkd1(-/-) embryos. In the kidneys of Pkd1(-/-), the expression of E-cadherin and PECAM in basolateral membranes of renal tubules was attenuated, and tyrosine phosphorylation of epidermal growth factor receptor and Gab1 were constitutively enhanced when cystogenesis started on embryonic day (E) 15.5-16.5. Maternally administered pioglitazone, a thiazolidinedione compound, resolved these molecular defects of Pkd1(-/-). Treatment with pioglitazone improved survival of Pkd1(-/-) embryos and ameliorated the cardiac defects and the degree of renal cystogenesis. Long-term treatment with pioglitazone improved the endothelial function of adult Pkd1(+/-). These data indicated that molecular defects observed in Pkd1(-/-) embryos contributed to the pathogenesis of ADPKD and that thiazolidinediones had a compensatory effect on the pathway affected by the loss of polycystin-1. Pathways activated by thiazolidinediones may provide new therapeutic targets in ADPKD.

Animals↗