Neuregulin-1 (NRG1) is thought to contribute to neuronal development and schizophrenia neuropatho... more Neuregulin-1 (NRG1) is thought to contribute to neuronal development and schizophrenia neuropathology. Recently, we have reported the animals treated with NRG1 as neonate later exhibit hyper-dopaminergic states and behavioral deficits related to schizophrenia, such as reduced social interaction and enhanced sensitivity to the psychostimulants (Kato et al., 2011. Mol. Psychiatry 16: 307-320). Though, NRG1 receptor (ErbB4) is highly enriched in dopaminergic neurons in the midbrain, it is unknown whether ErbB4 stimulation in this population influences on their electrophysiological features and synaptic properties. Here, we chronically treated with an EGF core domain peptide of NRG1 (eNRG1) as neonate, and analyzed spontaneous firing activity and electrophysiological properties of dopaminergic neurons in the adult midbrain slices. As a result, spontaneous firing activity was enhanced especially in the ventral tegmental area. Patch-clamp recording revealed increased frequencies of miniature EPSCs, which suggests potentiated excitatory synaptic transmission in this neuronal population. Currently, we are analyzing molecular mechanisms underlying these electrophysiological traits. The enhancement of spike firing rates and synaptic inputs might be associated with the behavioral deficits of the NRG1-treated animals.
Comparative sequence analyses have identified highly conserved genomic DNA sequences, including n... more Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENUinduced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results Electronic supplementary material The online version of this article (
ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human ... more ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human non-small cell lung cancer cases. More than 10 distinct ROS1-fusion genes, including break-point variants, have been identified to date. In this study, to investigate the in vivo oncogenic activities of one of the most frequently detected fusions, CD74-ROS1, as well as another SDC4-ROS1 fusion that has also been reported in several studies, we generated transgenic (TG) mouse strains that express either of the two ROS1-fusion genes specifically in lung alveolar type II cells. Mice in all TG lines developed tumorigenic nodules in the lung, and a few strains of both TG mouse lines demonstrated earlyonset nodule development (multiple tumor lesions present in the lung at 2-4 weeks after birth); therefore, these two strains were selected for further investigation. Tumors developed progressively in the untreated TG mice of both lines, whereas those receiving oral administration of an ALK/MET/ROS1 inhibitor, crizotinib, and an ALK/ROS1 inhibitor, ASP3026, showed marked reduction in the tumor burden. Collectively, these data suggest that each of these two ROS1fusion genes acts as a driver for the pathogenesis of lung adenocarcinoma in vivo. The TG mice developed in this study are expected to serve as valuable tools for exploring novel therapeutic agents against ROS1-fusion-positive lung cancer.
Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial ... more Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial complex II (MCII) has been recognized as a key component of HSCs. However, the physiological role of MCII on long-term hematopoiesis and hematopoietic reconstitution capacity remains unknown. Hence, this study evaluated the impact of MCII dysfunctions on long-term HSC maintenance and hematopoietic homeostasis among conditional transgenic mice with a missense mutation in the succinate dehydrogenase complex subunit C gene (SdhcV69E). HSCs collected from SdhcV69E mice had a higher reactive oxygen species (ROS) accumulation and DNA damage in response to mitochondrial activation. Via the aging stress response, MCII dysfunctions caused decreased white blood cell count with myeloid-skewing property, macrocytic anemia, and thrombocytosis. Moreover, the HSCs of aged SdhcV69E mice exhibited greater ROS accumulation and lower membrane potential. Transplantation-induced replicative stress also caused premature senescent hematopoiesis. Furthermore, accelerated ROS accumulation and profound DNA damage in HSCs were observed in the SdhcV69E-derived cell recipients. The long-term hematopoietic reconstitution capacity was remarkably impaired in HSCs from the SdhcV69E-derived cell recipients. Taken together, MCII plays an essential role in long-term hematopoiesis, and MCII dysfunctions with aging or replicative stresses caused excessive ROS accumulation and DNA damage in HSCs, leading to premature senescence.
It has become feasible to detect de novo mutations in mammalian genomes by using whole-genome seq... more It has become feasible to detect de novo mutations in mammalian genomes by using whole-genome sequencing. The power to detect numbers of de novo mutations should provide a useful tool to assess the transgenerational genetic effects of radiations on living organisms. By reviewing the spontaneous mutations in the mouse as a model, an action plan is proposed to detect the induced mutations after accumulating mutations for several generations with continuous exposure to low-dose radiations. Some susceptibility differences against radiations between humans and model animals for the transgenerational effect have been suggested. The applicability of the mouse model for the assessment of low-dose radiation is also discussed.
De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutati... more De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutations and the way they are inherited by somatic cells and germ cells remain unclear. Here, we present a novel method to reconstruct cell lineages. We identified mosaic mutations in mice using deep whole-genome sequencing and reconstructed embryonic cell lineages based on the variant allele frequencies of the mutations. The reconstructed trees were confirmed using nuclear transfer experiments and the genotyping of approximately 50 offspring of each tree. The most detailed tree had 32 terminal nodes and showed cell divisions from the fertilized egg to germ cell-and somatic cell-specific lineages, indicating at least five independent cell lineages that would be selected as founders of the primordial germ cells. The contributions of each lineage to germ cells and offspring varied widely. At the emergence of the germ cell-specific lineages, 10-15 embryonic mutations had accumulated, suggesting that the pregastrulation mutation rate is 1.0 mutation per mitosis. Subsequent mutation rates were 0.7 for germ cells and 13.2 for tail fibroblasts. Our results show a new framework to assess embryonic lineages; further, we suggest an evolutionary strategy for preserving heterogeneity owing to postzygotic mutations in offspring.
PurposePenile research is expected to reveal new targets for treatment and prevention of the comp... more PurposePenile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine.MethodsBy performing mouse N‐ethyl‐N‐nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism‐like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS)/MS analyses were performed.ResultsWe evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC–ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα.ConclusionsOur findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment.
Abstract To establish human disease models in the mouse, various forward and reverse genetics too... more Abstract To establish human disease models in the mouse, various forward and reverse genetics tools have been developed. New technologies, for example, high-throughput identification of novel mutations, gene targeting, and genome editing, in the mouse genome have made the forward and reverse genetics possible. Worldwide efforts including a variety of international consortiums have been producing many mutant mice with useful annotations for functional genomics to elucidate the mechanisms behind diseases. The resource centers and public databases have also been built to enhance their utilities. The modeling has started from Mendelian monogenic traits and has been expanding to more complex quantitative traits as well. Multidisciplinary integration among, for example, molecular, cellular and developmental biology, genetics, genomics, medicine, statistics, and informatics must be orchestrated to fully utilized the resources and knowledge of model mice.
Neuregulin-1 (NRG1) is thought to contribute to neuronal development and schizophrenia neuropatho... more Neuregulin-1 (NRG1) is thought to contribute to neuronal development and schizophrenia neuropathology. Recently, we have reported the animals treated with NRG1 as neonate later exhibit hyper-dopaminergic states and behavioral deficits related to schizophrenia, such as reduced social interaction and enhanced sensitivity to the psychostimulants (Kato et al., 2011. Mol. Psychiatry 16: 307-320). Though, NRG1 receptor (ErbB4) is highly enriched in dopaminergic neurons in the midbrain, it is unknown whether ErbB4 stimulation in this population influences on their electrophysiological features and synaptic properties. Here, we chronically treated with an EGF core domain peptide of NRG1 (eNRG1) as neonate, and analyzed spontaneous firing activity and electrophysiological properties of dopaminergic neurons in the adult midbrain slices. As a result, spontaneous firing activity was enhanced especially in the ventral tegmental area. Patch-clamp recording revealed increased frequencies of miniature EPSCs, which suggests potentiated excitatory synaptic transmission in this neuronal population. Currently, we are analyzing molecular mechanisms underlying these electrophysiological traits. The enhancement of spike firing rates and synaptic inputs might be associated with the behavioral deficits of the NRG1-treated animals.
Comparative sequence analyses have identified highly conserved genomic DNA sequences, including n... more Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENUinduced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results Electronic supplementary material The online version of this article (
ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human ... more ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human non-small cell lung cancer cases. More than 10 distinct ROS1-fusion genes, including break-point variants, have been identified to date. In this study, to investigate the in vivo oncogenic activities of one of the most frequently detected fusions, CD74-ROS1, as well as another SDC4-ROS1 fusion that has also been reported in several studies, we generated transgenic (TG) mouse strains that express either of the two ROS1-fusion genes specifically in lung alveolar type II cells. Mice in all TG lines developed tumorigenic nodules in the lung, and a few strains of both TG mouse lines demonstrated earlyonset nodule development (multiple tumor lesions present in the lung at 2-4 weeks after birth); therefore, these two strains were selected for further investigation. Tumors developed progressively in the untreated TG mice of both lines, whereas those receiving oral administration of an ALK/MET/ROS1 inhibitor, crizotinib, and an ALK/ROS1 inhibitor, ASP3026, showed marked reduction in the tumor burden. Collectively, these data suggest that each of these two ROS1fusion genes acts as a driver for the pathogenesis of lung adenocarcinoma in vivo. The TG mice developed in this study are expected to serve as valuable tools for exploring novel therapeutic agents against ROS1-fusion-positive lung cancer.
Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial ... more Mitochondria are indispensable in maintaining hematopoietic stem cells (HSCs), and mitochondrial complex II (MCII) has been recognized as a key component of HSCs. However, the physiological role of MCII on long-term hematopoiesis and hematopoietic reconstitution capacity remains unknown. Hence, this study evaluated the impact of MCII dysfunctions on long-term HSC maintenance and hematopoietic homeostasis among conditional transgenic mice with a missense mutation in the succinate dehydrogenase complex subunit C gene (SdhcV69E). HSCs collected from SdhcV69E mice had a higher reactive oxygen species (ROS) accumulation and DNA damage in response to mitochondrial activation. Via the aging stress response, MCII dysfunctions caused decreased white blood cell count with myeloid-skewing property, macrocytic anemia, and thrombocytosis. Moreover, the HSCs of aged SdhcV69E mice exhibited greater ROS accumulation and lower membrane potential. Transplantation-induced replicative stress also caused premature senescent hematopoiesis. Furthermore, accelerated ROS accumulation and profound DNA damage in HSCs were observed in the SdhcV69E-derived cell recipients. The long-term hematopoietic reconstitution capacity was remarkably impaired in HSCs from the SdhcV69E-derived cell recipients. Taken together, MCII plays an essential role in long-term hematopoiesis, and MCII dysfunctions with aging or replicative stresses caused excessive ROS accumulation and DNA damage in HSCs, leading to premature senescence.
It has become feasible to detect de novo mutations in mammalian genomes by using whole-genome seq... more It has become feasible to detect de novo mutations in mammalian genomes by using whole-genome sequencing. The power to detect numbers of de novo mutations should provide a useful tool to assess the transgenerational genetic effects of radiations on living organisms. By reviewing the spontaneous mutations in the mouse as a model, an action plan is proposed to detect the induced mutations after accumulating mutations for several generations with continuous exposure to low-dose radiations. Some susceptibility differences against radiations between humans and model animals for the transgenerational effect have been suggested. The applicability of the mouse model for the assessment of low-dose radiation is also discussed.
De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutati... more De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutations and the way they are inherited by somatic cells and germ cells remain unclear. Here, we present a novel method to reconstruct cell lineages. We identified mosaic mutations in mice using deep whole-genome sequencing and reconstructed embryonic cell lineages based on the variant allele frequencies of the mutations. The reconstructed trees were confirmed using nuclear transfer experiments and the genotyping of approximately 50 offspring of each tree. The most detailed tree had 32 terminal nodes and showed cell divisions from the fertilized egg to germ cell-and somatic cell-specific lineages, indicating at least five independent cell lineages that would be selected as founders of the primordial germ cells. The contributions of each lineage to germ cells and offspring varied widely. At the emergence of the germ cell-specific lineages, 10-15 embryonic mutations had accumulated, suggesting that the pregastrulation mutation rate is 1.0 mutation per mitosis. Subsequent mutation rates were 0.7 for germ cells and 13.2 for tail fibroblasts. Our results show a new framework to assess embryonic lineages; further, we suggest an evolutionary strategy for preserving heterogeneity owing to postzygotic mutations in offspring.
PurposePenile research is expected to reveal new targets for treatment and prevention of the comp... more PurposePenile research is expected to reveal new targets for treatment and prevention of the complex mechanisms of its disorder including erectile dysfunction (ED). Thus, analyses of the molecular processes of penile ED and continuous erection as priapism are essential issues of reproductive medicine.MethodsBy performing mouse N‐ethyl‐N‐nitrosourea mutagenesis and exome sequencing, we established a novel mouse line displaying protruded genitalia phenotype (PGP; priapism‐like phenotype) and identified a novel Pitpna gene mutation for PGP. Extensive histological analyses on the Pitpna mutant and intracavernous pressure measurement (ICP) and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI/MS)/MS analyses were performed.ResultsWe evaluated the role of phospholipids during erection for the first time and showed the mutants of inducible phenotypes of priapism. Moreover, quantitative analysis using LC–ESI/MS/MS revealed that the level of phosphatidylinositol (PI) was significantly lower in the mutant penile samples. These results imply that PI may contribute to penile erection by PITPα.ConclusionsOur findings suggest that the current mutant is a mouse model for priapism and abnormalities in PI signaling pathways through PITPα may lead to priapism providing an attractive novel therapeutic target in its treatment.
Abstract To establish human disease models in the mouse, various forward and reverse genetics too... more Abstract To establish human disease models in the mouse, various forward and reverse genetics tools have been developed. New technologies, for example, high-throughput identification of novel mutations, gene targeting, and genome editing, in the mouse genome have made the forward and reverse genetics possible. Worldwide efforts including a variety of international consortiums have been producing many mutant mice with useful annotations for functional genomics to elucidate the mechanisms behind diseases. The resource centers and public databases have also been built to enhance their utilities. The modeling has started from Mendelian monogenic traits and has been expanding to more complex quantitative traits as well. Multidisciplinary integration among, for example, molecular, cellular and developmental biology, genetics, genomics, medicine, statistics, and informatics must be orchestrated to fully utilized the resources and knowledge of model mice.
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Papers by Yoichi Gondo