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1.
Cell Rep ; 2(5): 1363-74, 2012 Nov 29.
Article in English | MEDLINE | ID: mdl-23142660

ABSTRACT

After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entrée to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.


Subject(s)
Embryonic Stem Cells/cytology , Proteins/metabolism , t-Complex Genome Region , Animals , Base Sequence , Cell Communication , Cell Differentiation , Cells, Cultured , Ectoderm/metabolism , Ectoderm/pathology , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Embryonic Development , Embryonic Stem Cells/metabolism , Exons , Frameshift Mutation , Haplotypes , Major Histocompatibility Complex/genetics , Mice , Mice, Knockout , Mice, Transgenic , Mitosis , Molecular Sequence Data , Proteins/genetics , Vesicular Transport Proteins
2.
Eur J Neurosci ; 33(2): 205-13, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21105914

ABSTRACT

Since the discovery of odorant receptors (ORs) in rodents, most ORs have remained orphan receptors. Even for deorphanized ORs in vitro, their in vivo properties are largely unknown. Here, we report odor response profiles of two highly homologous mouse ORs, MOR29A and MOR29B, both in vivo and in vitro. The BAC transgenic mouse was generated, in which olfactory sensory neurons (OSNs) expressing the transgenes MOR29A and MOR29B were differently tagged with IRES-gapECFP and IRES-gapEYFP, respectively. MOR29A- and MOR29B-expressing OSN axons converged on separate but nearby loci on the dorsal surface of the olfactory bulb (OB). Optical imaging of intrinsic signals in the OB identified five different phenyl ethers as candidate ligands for MOR29B. Based on in vitro calcium imaging with the isolated OSNs and luciferase assay with heterologous cells, only guaiacol and vanillin were found to be potent agonists for MOR29A and MOR29B. Because of its accessible glomerular locations in the dorsal OB and defined odor response profiles both in vivo and in vitro, the MOR29A/29B tagging mouse will serve as an excellent tool for studying both odor-signal processing and neural circuitry in the OB.


Subject(s)
Olfactory Receptor Neurons/metabolism , Phenyl Ethers/metabolism , Receptors, Odorant/metabolism , Animals , Benzaldehydes/chemistry , Benzaldehydes/metabolism , Cell Line , Dose-Response Relationship, Drug , Guaiacol/chemistry , Guaiacol/metabolism , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Molecular Structure , Olfactory Bulb/anatomy & histology , Olfactory Pathways/metabolism , Olfactory Receptor Neurons/cytology , Phenyl Ethers/chemistry , Receptors, Odorant/genetics , Transgenes
3.
Cell ; 141(6): 1056-67, 2010 Jun 11.
Article in English | MEDLINE | ID: mdl-20550939

ABSTRACT

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) roughly correlate with their axonal projection sites along the dorsal-ventral (D-V) axis of the olfactory bulb (OB). Here we report that an axon guidance receptor, Neuropilin-2 (Nrp2), and its repulsive ligand, Semaphorin-3F (Sema3F), are expressed by OSNs in a complementary manner that is important for establishing olfactory map topography. Sema3F is secreted by early-arriving axons of OSNs and is deposited at the anterodorsal OB to repel Nrp2-positive axons that arrive later. Sequential arrival of OSN axons as well as the graded and complementary expression of Nrp2 and Sema3F by OSNs help to form the topographic order along the D-V axis.


Subject(s)
Axons/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Olfactory Bulb/metabolism , Animals , Gene Expression , Membrane Proteins/genetics , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Neuropilin-2/metabolism , Receptors, Cell Surface/metabolism , X Chromosome Inactivation
4.
J Immunol ; 184(10): 5874-84, 2010 May 15.
Article in English | MEDLINE | ID: mdl-20393137

ABSTRACT

Many members of the nucleotide-binding and oligomerization domain (NOD)- and leucine-rich-repeat-containing protein (NLR) family play important roles in pathogen recognition and inflammation. However, we previously reported that human PYNOD/NLRP10, an NLR-like protein consisting of a pyrin domain and a NOD, inhibits inflammatory signal mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in reconstitution experiments using HEK293 cells. In this study, we investigated the molecular mechanism of PYNOD's anti-inflammatory activity in vitro and its expression and function in mice. Human PYNOD inhibited the autoprocessing of caspase-1 and caspase-1-mediated IL-1beta processing and suppressed the aggregation of ASC, a hallmark of ASC activation. Interestingly, the NOD of human PYNOD was sufficient to inhibit caspase-1-mediated IL-1beta secretion, whereas its pyrin domain was sufficient to inhibit ASC-mediated NF-kappaB activation and apoptosis and to reduce ASC's ability to promote caspase-1-mediated IL-1beta production. Mouse PYNOD protein was detected in the skin, tongue, heart, colon, peritoneal macrophages, and several cell lines of hematopoietic and myocytic lineages. Mouse PYNOD colocalized with ASC aggregates in LPS + R837-stimulated macrophages; however, unlike human PYNOD, mouse PYNOD failed to inhibit ASC aggregation. Macrophages and neutrophils from PYNOD-transgenic mice exhibited reduced IL-1beta processing and secretion upon microbial infection, although mouse PYNOD failed to inhibit caspase-1 processing, which was inhibited by caspase-4 inhibitor z-LEED-fluoromethylketone. These results suggest that mouse PYNOD colocalizes with ASC and inhibits caspase-1-mediated IL-1beta processing without inhibiting caspase-4 (mouse caspase-11)-mediated caspase-1 processing. Furthermore, PYNOD-transgenic mice were resistant to lethal endotoxic shock. Thus, PYNOD is the first example of an NLR that possesses an anti-inflammatory function in vivo.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Carrier Proteins/physiology , Inflammation Mediators/physiology , Adaptor Proteins, Signal Transducing , Animals , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/genetics , Apoptosis/immunology , Apoptosis Regulatory Proteins , CARD Signaling Adaptor Proteins , Carrier Proteins/genetics , Carrier Proteins/metabolism , Caspase Inhibitors , Caspases/physiology , Caspases, Initiator , Cell Line , Cell Line, Tumor , Cells, Cultured , Cytokines/biosynthesis , Cytoskeletal Proteins/blood , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Cytoskeletal Proteins/physiology , Enzyme Precursors/antagonists & inhibitors , Humans , Inflammation Mediators/metabolism , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/blood , Lipopolysaccharides/toxicity , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Shock, Septic/immunology , Shock, Septic/prevention & control , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/blood
5.
Science ; 325(5940): 585-90, 2009 Jul 31.
Article in English | MEDLINE | ID: mdl-19589963

ABSTRACT

Sensory information detected by the peripheral nervous system is represented as a topographic map in the brain. It has long been thought that the topography of the map is determined by graded positional cues that are expressed by the target. Here, we analyzed the pre-target axon sorting for olfactory map formation in mice. In olfactory sensory neurons, an axon guidance receptor, Neuropilin-1, and its repulsive ligand, Semaphorin-3A, are expressed in a complementary manner. We found that expression levels of Neuropilin-1 determined both pre-target sorting and projection sites of axons. Olfactory sensory neuron-specific knockout of Semaphorin-3A perturbed axon sorting and altered the olfactory map topography. Thus, pre-target axon sorting plays an important role in establishing the topographic order based on the relative levels of guidance molecules expressed by axons.


Subject(s)
Axons/physiology , Neuropilin-1/metabolism , Olfactory Bulb/physiology , Olfactory Pathways/physiology , Olfactory Receptor Neurons/physiology , Animals , Brain Mapping , Cell Communication , Cues , Cyclic AMP/metabolism , Ligands , Mice , Mice, Knockout , Mice, Transgenic , Neuroglia/physiology , Olfactory Bulb/cytology , Olfactory Mucosa/cytology , Olfactory Mucosa/physiology , Olfactory Pathways/cytology , Olfactory Receptor Neurons/cytology , Receptors, Odorant/metabolism , Semaphorin-3A/metabolism , Signal Transduction
6.
Transgenic Res ; 17(5): 817-26, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18357510

ABSTRACT

Transthyretin (TTR) associated amyloidosis is an autosomal dominant disorder characterized by peripheral and autonomic neuropathy. Both genetic and environmental factors are thought to be involved in development of TTR associated amyloidosis. Previously, we demonstrated that amyloid deposition was observed in various tissues of transgenic mouse lines carrying a human mutant TTR (Met30) gene. To analyze the influence of environmental factors on TTR amyloidosis, these amyloidogenic transgenic mouse models were kept under conventional (CV) or specific pathogen free (SPF) conditions. Although the serum levels of Met30 for mice housed in the CV and SPF conditions were similar, amyloid deposition was observed in CV conditions, but not in SPF conditions. In addition, the extent of amyloid deposition in transgenic mice was dependent on duration kept under CV conditions. There were significant differences in proportion of amyloid deposition in several tissues between CV and SPF conditions. Maintenance of these mice at 30 degrees C did not induce amyloid deposition in SPF conditions. These results suggest that the SPF conditions can completely prevent amyloid deposition, and that environmental factors can affect the onset and progression even in a single gene disorder.


Subject(s)
Amyloidosis/prevention & control , Disease Models, Animal , Prealbumin/metabolism , Amyloidosis/metabolism , Animals , Blotting, Western , Mice , Polymerase Chain Reaction
7.
Proc Natl Acad Sci U S A ; 105(3): 1003-8, 2008 Jan 22.
Article in English | MEDLINE | ID: mdl-18192401

ABSTRACT

Infection with cagA-positive Helicobacter pylori is associated with gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) lymphoma of B cell origin. The cagA-encoded CagA protein is delivered into gastric epithelial cells via the bacterial type IV secretion system and, upon tyrosine phosphorylation by Src family kinases, specifically binds to and aberrantly activates SHP-2 tyrosine phosphatase, a bona fide oncoprotein in human malignancies. CagA also elicits junctional and polarity defects in epithelial cells by interacting with and inhibiting partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) independently of CagA tyrosine phosphorylation. Despite these CagA activities that contribute to neoplastic transformation, a causal link between CagA and in vivo oncogenesis remains unknown. Here, we generated transgenic mice expressing wild-type or phosphorylation-resistant CagA throughout the body or predominantly in the stomach. Wild-type CagA transgenic mice showed gastric epithelial hyperplasia and some of the mice developed gastric polyps and adenocarcinomas of the stomach and small intestine. Systemic expression of wild-type CagA further induced leukocytosis with IL-3/GM-CSF hypersensitivity and some mice developed myeloid leukemias and B cell lymphomas, the hematological malignancies also caused by gain-of-function SHP-2 mutations. Such pathological abnormalities were not observed in transgenic mice expressing phosphorylation-resistant CagA. These results provide first direct evidence for the role of CagA as a bacterium-derived oncoprotein (bacterial oncoprotein) that acts in mammals and further indicate the importance of CagA tyrosine phosphorylation, which enables CagA to deregulate SHP-2, in the development of H. pylori-associated neoplasms.


Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Gastrointestinal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Helicobacter pylori/metabolism , Hematologic Neoplasms/metabolism , Animals , Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Cell Transformation, Neoplastic/genetics , Gastrointestinal Neoplasms/genetics , Gastrointestinal Neoplasms/pathology , Helicobacter pylori/genetics , Helicobacter pylori/pathogenicity , Hematologic Neoplasms/genetics , Hematologic Neoplasms/pathology , Mice , Mice, Transgenic , Phosphotyrosine/metabolism
10.
Mol Genet Metab ; 91(3): 285-93, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17482496

ABSTRACT

Calreticulin (CRT) is a Ca(2+)-binding protein of the endoplasmic reticulum essential for cardiac development. For further investigation of the functional mechanism of calreticulin, we generated transgenic mice with spatiotemporal overexpression of calreticulin using a cre-loxP system. To elucidate the role of the protein in cardiogenesis, we adopted Nkx2.5-cre mice for heart specific overexpression. The overexpression of calreticulin was associated with arrhythmia, chamber dilation and sudden death, as observed in 6- to 10-week-old mice. Furthermore, transgenic mice displayed marked edema at 7-weeks of age. RT-PCR analysis revealed that the expression of hyperpolerization-activated cyclic nucleotide-gated channel1 (HCN1), an essential component for cardiac pace maker activity, had receded in the heart of transgenic mice. In addition, the protein level of connexin40 (Cx40), connexin43 (Cx43), components of gap junction, and myocyte-enhancer factor (MEF) 2C, a cardiac-specific transcriptional factor, were reduced in the transgenic mice hearts. These findings suggest that calreticulin affects cardiac arrhythmia with disruption of cardiac signaling, such as the HCN family members, and with low levels of Cx40 and Cx43. Overepression of calreticulin also leads to a decreased protein level of MEF2C and this may cause changes in cardiac structure. Our findings support calreticulin being critical for normal heart function and structure. These mice are a useful model for the study of endoplasmic reticulum proteins, such as calreticulin, in various tissues.


Subject(s)
Arrhythmias, Cardiac/metabolism , Calreticulin/metabolism , Animals , Arrhythmias, Cardiac/pathology , Arrhythmias, Cardiac/physiopathology , Calreticulin/genetics , Connexin 43/metabolism , Connexins/metabolism , Cyclic Nucleotide-Gated Cation Channels , Endoplasmic Reticulum/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , MEF2 Transcription Factors , Mice , Mice, Transgenic , Myocardium/metabolism , Myocardium/pathology , Myogenic Regulatory Factors/metabolism , Potassium Channels/metabolism , Gap Junction alpha-5 Protein
11.
Hepatol Res ; 37(2): 133-42, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17300709

ABSTRACT

AIM: Apoptosis is involved in hepatic ischemia/reperfusion injury. The protein FNK, constructed from an anti-apoptotic protein Bcl-x(L), exhibits the stronger anticell death activity. We evaluated the effect of FNK on apoptosis after hepatic ischemia and reperfusion, using FNK-overexpressing transgenic mice and the HIV/Tat protein-transduction-domain (PTD) that mediates the introduction of FNK into cells when fused with FNK (PTD-FNK). METHODS: Mice were given hepatic ischemic insult for 90 min followed by reperfusion for 3 h. FNK overexpression was determined by immunohistochemistry and Western blot. PTD-FNK was intraperitoneally injected into wild mice 3 h before the insult. Liver injury was determined by the caspase activation, DNA fragmentation, and hematoxylin-eosin and terminal deoxynucleotidyl transferase-mediated dUTP- digoxigenin nick-end labelling (TUNEL) stainings. RESULTS: In FNK-transgenic mice, FNK overexpression inhibited the activation of caspase 3/caspase 3-like activity and DNA fragmentation caused by the injury. In wild mice preinjected with PTD-FNK, PTD-FNK significantly inhibited the caspase activation and DNA fragmentation, reduced the area of liver vacuolization, and protected hepatic cells surrounding blood vessels, irrespective of central or portal veins, from the ischemia/reperfusion damage. CONCLUSIONS: FNK inhibits apoptotic death due to the ischemia/reperfusion injury. Our results provide the reasonable expectation of therapeutic protein PTD-FNK for clinical applications, such as transplantation, to protect against ischemia/reperfusion injury.

12.
Am J Physiol Cell Physiol ; 292(6): C2141-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17215327

ABSTRACT

Defensins comprise a family of cationic antimicrobial peptides characterized by conserved cysteine residues. They are produced in various organs including skeletal muscle and are identified as key elements in the host defense system as potent effectors. At the same time, defensins have potential roles in the regulation of inflammation and, furthermore, can exert cytotoxic effects on several mammalian cells. Here, we developed transgenic mice overexpressing mouse beta-defensin-6 to explore the pathophysiological roles of the defensin family as a novel mediator of inflammatory tissue injury. Unexpectedly, the transgenic mice showed short lifespan, poor growth, and progressive myofiber degeneration with functional muscle impairment, predominant centronucleated myofibers, and elevated serum creatine kinase activity, as seen in human muscular dystrophy. Furthermore, some of the transgenic myofibers showed IkappaBalpha accumulation, which would be related to the myofiber apoptosis of limb-girdle muscular dystrophy type 2A. The present findings may unravel a concealed linkage between the innate immune system and the pathophysiology of degenerative diseases.


Subject(s)
Defensins/metabolism , Muscle, Skeletal/metabolism , Muscular Atrophy/genetics , Muscular Atrophy/metabolism , Aging , Animals , Defensins/genetics , Female , Gene Expression Regulation , I-kappa B Kinase/metabolism , Kyphosis , Longevity , Male , Mice , Mice, Transgenic , Muscle, Skeletal/pathology , Weight Gain
13.
Cell ; 127(5): 1057-69, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17129788

ABSTRACT

In the mouse, olfactory sensory neurons (OSNs) expressing the same odorant receptor (OR) converge their axons to a specific set of glomeruli in the olfactory bulb. To study how OR-instructed axonal fasciculation is controlled, we searched for genes whose expression profiles are correlated with the expressed ORs. Using the transgenic mouse in which the majority of OSNs express a particular OR, we identified such genes coding for the homophilic adhesive molecules Kirrel2/Kirrel3 and repulsive molecules ephrin-A5/EphA5. In the CNGA2 knockout mouse, where the odor-evoked cation influx is disrupted, Kirrel2 and EphA5 were downregulated, while Kirrel3 and ephrin-A5 were upregulated, indicating that these genes are transcribed in an activity-dependent manner. Mosaic analysis demonstrated that gain of function of these genes generates duplicated glomeruli. We propose that a specific set of adhesive/repulsive molecules, whose expression levels are determined by OR molecules, regulate the axonal fasciculation of OSNs during the process of glomerular map formation.


Subject(s)
Axons/metabolism , Neurons, Afferent/cytology , Neurons, Afferent/metabolism , Receptors, Odorant/metabolism , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Adhesion Molecules/metabolism , Ephrin-A5/genetics , Ephrin-A5/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Transgenic , Models, Biological , Olfactory Bulb/cytology , Protein Binding , Receptor, EphA5/metabolism , Receptors, Odorant/genetics
14.
Science ; 314(5799): 657-61, 2006 Oct 27.
Article in English | MEDLINE | ID: mdl-16990513

ABSTRACT

In mammals, odorant receptors (ORs) direct the axons of olfactory sensory neurons (OSNs) toward targets in the olfactory bulb. We show that cyclic adenosine monophosphate (cAMP) signals that regulate the expression of axon guidance molecules are essential for the OR-instructed axonal projection. Genetic manipulations of ORs, stimulatory G protein, cAMP-dependent protein kinase, and cAMP response element-binding protein shifted the axonal projection sites along the anteriorposterior axis in the olfactory bulb. Thus, it is the OR-derived cAMP signals, rather than direct action of OR molecules, that determine the target destinations of OSNs.


Subject(s)
Axons/physiology , Cyclic AMP/metabolism , Olfactory Bulb/physiology , Olfactory Receptor Neurons/physiology , Receptors, Odorant/metabolism , Signal Transduction , Animals , Animals, Genetically Modified , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation , Neuropilin-1/genetics , Neuropilin-1/metabolism , Olfactory Bulb/cytology , Oligonucleotide Array Sequence Analysis , Rats , Rats, Wistar , Transcription, Genetic
15.
Hum Mol Genet ; 15(14): 2266-75, 2006 Jul 15.
Article in English | MEDLINE | ID: mdl-16777842

ABSTRACT

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle-wasting disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin. Therapeutic options for DMD are limited because the pathogenetic mechanism by which dystrophin deficiency produces the clinical phenotype remains obscure. Recent reports of abnormal alpha-adrenergic vasoregulation in the exercising muscles of DMD patients and in the mdx mouse, an animal model of DMD, prompted us to hypothesize that the dystrophin-deficient smooth muscle contributes to the vascular and dystrophic phenotypes of DMD. To test this, we generated transgenic mdx mice that express dystrophin only in smooth muscle (SMTg/mdx). We found that alpha-adrenergic vasoconstriction was markedly attenuated in the contracting hindlimbs of C57BL/10 wild-type mice, an effect that was mediated by nitric oxide (NO) and was severely impaired in the mdx mice. SMTg/mdx mice showed an intermediate phenotype, with partial restoration of the NO-dependent modulation of alpha-adrenergic vasoconstriction in active muscle. In addition, the elevated serum creatine kinase levels observed in mdx mice were significantly reduced in SMTg/mdx mice. This is the first report of a functional role of dystrophin in vascular smooth muscle.


Subject(s)
Dystrophin/genetics , Dystrophin/physiology , Muscle, Smooth, Vascular/physiopathology , Muscular Dystrophy, Animal/genetics , Muscular Dystrophy, Animal/physiopathology , Animals , Base Sequence , Creatine Kinase/blood , DNA, Complementary/genetics , Female , Gene Expression , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Transgenic , Muscle Contraction , Muscle, Smooth, Vascular/blood supply , Muscular Dystrophy, Animal/pathology , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/physiopathology , Nitric Oxide Synthase/antagonists & inhibitors , Receptors, Adrenergic, alpha/physiology , Vasoconstriction
16.
Genetics ; 172(1): 445-56, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16204209

ABSTRACT

We established the mutant mouse line, B6;CB-SktGtAyu8021IMEG (SktGt), through gene-trap mutagenesis in embryonic stem cells. The novel gene identified, called Sickle tail (Skt), is composed of 19 exons and encodes a protein of 1352 amino acids. Expression of a reporter gene was detected in the notochord during embryogenesis and in the nucleus pulposus of mice. Compression of some of the nuclei pulposi in the intervertebral discs (IVDs) appeared at embryonic day (E) 17.5, resulting in a kinky-tail phenotype showing defects in the nucleus pulposus and annulus fibrosus of IVDs in SktGt/Gt mice. These phenotypes were different from those in Danforth's short tail (Sd) mice in which the nucleus pulposus was totally absent and replaced by peripheral fibers similar to those seen in the annulus fibrosus in all IVDs. The Skt gene maps to the proximal part of mouse chromosome 2, near the Sd locus. The genetic distance between them was 0.95 cM. The number of vertebrae in both [Sd +/+ SktGt] and [Sd SktGt/+ +] compound heterozygotes was less than that of Sd heterozygotes. Furthermore, the enhancer trap locus Etl4lacZ, which was previously reported to be an allele of Sd, was located in the third intron of the Skt gene.


Subject(s)
Fetal Development/genetics , Intervertebral Disc/abnormalities , Mice/genetics , Mutation , Proteins/physiology , Tail/abnormalities , Amino Acid Sequence , Animals , Base Sequence , Cell Nucleus/genetics , Cloning, Molecular , Crosses, Genetic , Exons/genetics , Female , Gene Expression Regulation, Developmental , Male , Mice/embryology , Mice, Inbred C57BL , Molecular Sequence Data , Mutagenesis , Notochord/abnormalities , Notochord/embryology , Proteins/genetics , Stem Cells/metabolism , Tail/embryology , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
17.
Dev Dyn ; 232(2): 282-92, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15614767

ABSTRACT

Sonic hedgehog (Shh) is a secreted molecule that is thought to regulate tissue growth and patterning in vertebrate embryos. Although it has been reported that Gli transcription factors mediate Shh signaling to the nucleus, little is known about developmental target genes of Gli. In the previous genetic study, we showed that Shh is required for Fgf15 expression in the diencephalon and midbrain. Here, we examined whether Fgf15 is a direct target of Shh signaling through Gli. Shh was expressed in the midline cells and Fgf15 in the medial region of the diencephalon/midbrain by the seven-somite stage. The Fgf15 expression domain coincided with that of Gli1 and overlapped with that of Gli2 at this stage. Fgf15 expression in the diencephalon/midbrain was greatly reduced in the seven-somite Shh mutant embryos. Transgenic analysis showed that the 3.6-kb 5'-flanking region of the Fgf15 gene is sufficient for induction of Fgf15 in the medial/ventral diencephalon/midbrain. Luciferase assay showed that the 3.6-kb Fgf15 enhancer/promoter was activated by Gli2. A Gli-binding site was located 1 kb upstream of the transcription start site and was required for expression in the medial/ventral diencephalon/midbrain in transgenic embryos and for activation in luciferase assay. These findings indicate that Fgf15 is directly regulated by Shh signaling through Gli proteins.


Subject(s)
Diencephalon/embryology , Fibroblast Growth Factors/biosynthesis , Gene Expression Regulation, Developmental , Mesencephalon/embryology , Oncogene Proteins/biosynthesis , Trans-Activators/metabolism , Transcription Factors/biosynthesis , Amino Acid Motifs , Animals , Base Sequence , Binding Sites , Brain/metabolism , Cell Line , Cell Nucleus/metabolism , Cloning, Molecular , Enhancer Elements, Genetic , Genes, Reporter , Glutathione Transferase/metabolism , Hedgehog Proteins , In Situ Hybridization , Kruppel-Like Transcription Factors , Luciferases/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Models, Genetic , Molecular Sequence Data , Mutagenesis , Mutation , Nucleic Acid Hybridization , Plasmids/metabolism , Promoter Regions, Genetic , Protein Structure, Tertiary , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transcription, Genetic , Transgenes , Zinc Finger Protein GLI1 , Zinc Finger Protein Gli2 , beta-Galactosidase/metabolism
18.
Oncogene ; 23(54): 8720-30, 2004 Nov 18.
Article in English | MEDLINE | ID: mdl-15480417

ABSTRACT

Aurora-A, a serine/threonine mitotic kinase, was reported to be overexpressed in various human cancers, and its overexpression induces aneuploidy, centrosome amplification and tumorigenic transformation in cultured human and rodent cells. However, the underlying mechanisms and pathological settings by which Aurora-A promotes tumorigenesis are largely unknown. Here, we created a transgenic mouse model to investigate the involvement of Aurora-A overexpression in the development of mammary glands and tumorigenesis using a Cre-loxP system. The conditional expression of Aurora-A resulted in significantly increased binucleated cell formation and apoptosis in the mammary epithelium. The surviving mammary epithelial cells composed hyperplastic areas after a short latency. Induction of Aurora-A overexpression in mouse embryonic fibroblasts prepared from the transgenic mice also led to aberrant mitosis and binucleated cell formation followed by apoptosis. The levels of p53 protein were remarkably increased in these Aurora-A-overexpressing cells, and the apoptosis was significantly suppressed by deletion of p53. Given that no malignant tumor formation was found in the Aurora-A-overexpressing mouse model after a long latency, additional factors, such as p53 inactivation, are required for the tumorigenesis of Aurora-A-overexpressing mammary epithelium. Our findings indicated that this mouse model is a useful system to study the physiological roles of Aurora-A and the genetic pathways of Aurora-A-induced carcinogenesis.


Subject(s)
Mammary Glands, Animal/pathology , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Recombinases/physiology , Animals , Apoptosis/physiology , Aurora Kinase A , Aurora Kinases , Base Sequence , Cell Transformation, Neoplastic , Fluorescent Antibody Technique , Hyperplasia , Mammary Neoplasms, Experimental/enzymology , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Models, Animal , Molecular Sequence Data , Protein Serine-Threonine Kinases/genetics , Recombinases/metabolism , Tumor Suppressor Protein p53/physiology
19.
Mol Cell Biol ; 24(18): 8007-17, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15340063

ABSTRACT

Aberrant protein folding beyond the capacity of endoplasmic reticulum (ER) quality control leads to stress response in the ER. The Lys-Asp-Glu-Leu (KDEL) receptor, a retrieval receptor for ER chaperones in the early secretory pathway, contributes to ER quality control. To elucidate the function of the KDEL receptor in vivo, we established transgenic mice expressing a mutant KDEL receptor. We found that the mutant KDEL receptor sensitized cells to ER stress and that the mutant mice developed dilated cardiomyopathy. Ultrastructural analyses revealed expanded sarcoplasmic reticulums and protein aggregates that obstructed the adjacent transverse tubules of the mutant cardiomyocytes. Cardiomyocytes from the mutant mice were sensitive to ER stress when treated with tunicamycin and showed a functional defect in the L-type Ca(2+) current. We observed ubiquitinated protein aggregates, enhanced expression of CHOP (a death-related transcriptional factor expressed upon ER stress), and apoptosis in the mutant hearts. These findings suggest that impairment of the KDEL receptor disturbs ER quality control, resulting in accumulation of misfolded proteins in the ER in an in vivo system, and that the dilated cardiomyopathy found in the mutant KDEL receptor transgenic mice is associated with ER stress.


Subject(s)
Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/metabolism , Endoplasmic Reticulum/metabolism , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Animals , Calcium Signaling , Cardiomyopathy, Dilated/etiology , Cardiomyopathy, Dilated/pathology , Golgi Apparatus/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Models, Cardiovascular , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/ultrastructure , Protein Folding , Rats , Rats, Sprague-Dawley , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum/ultrastructure , Stress, Mechanical
20.
J Immunol ; 173(7): 4675-83, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15383603

ABSTRACT

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructural protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-alpha increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2(b) haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.


Subject(s)
Arthritis, Experimental/genetics , Arthritis, Experimental/virology , Genetic Predisposition to Disease , Mice, Transgenic/virology , Parvovirus B19, Human/genetics , Viral Nonstructural Proteins/genetics , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/virology , Autoantibodies/blood , Base Sequence , Collagen Type II/administration & dosage , Collagen Type II/immunology , Crosses, Genetic , Female , Humans , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymph Nodes/virology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Molecular Sequence Data , Parvovirus B19, Human/immunology , Synovial Membrane/pathology , Synovial Membrane/virology , Tumor Necrosis Factor-alpha/metabolism , Viral Nonstructural Proteins/administration & dosage
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