Microstripe pattern substrate consisting of alternating planar and nanoprotrusive regions improved hiPSC-derived cardiomyocytes' unidirectional alignment and functional properties.

The alignment of each cell in human myocardium is considered critical for the efficient movement of cardiac tissue. We investigated 96-well microstripe-patterned plates to align human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), which resemble fetal myocardium. The aligned CMs (ACMs) cultured on the microstripe-patterned plates exhibited pathology, motor function, gene expression, and drug response that more closely resembled those of adult cells than did unaligned CMs cultured on a flat plate (FCMs). We used these ACMs to evaluate drug side effects and efficacy, and to determine whether these were similar to adult-like responses. When CMs from patients with hypertrophic cardiomyopathy (HCMs) were seeded and cultured on the microstripe-patterned plates or layered on top of the ACMs, both sets of HCMs showed increased heart rate and synchronized contractions, indicating improved cardiac function. It is suggested that the ACMs could be used for drug screening as cells representative of adult-like CMs and be transplanted in the form of a cell sheet for regenerative treatment of heart failure.

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.

In Vitro Models of GJB2-Related Hearing Loss Recapitulate Ca2+ Transients via a Gap Junction Characteristic of Developing Cochlea.

Mutation of the Gap Junction Beta 2 gene (GJB2) encoding connexin 26 (CX26) is the most frequent cause of hereditary deafness worldwide and accounts for up to 50% of non-syndromic sensorineural hearing loss cases in some populations. Therefore, cochlear CX26-gap junction plaque (GJP)-forming cells such as cochlear supporting cells are thought to be the most important therapeutic target for the treatment of hereditary deafness. The differentiation of pluripotent stem cells into cochlear CX26-GJP-forming cells has not been reported. Here, we detail the development of a novel strategy to differentiate induced pluripotent stem cells into functional CX26-GJP-forming cells that exhibit spontaneous ATP- and hemichannel-mediated Ca2+ transients typical of the developing cochlea. Furthermore, these cells from CX26-deficient mice recapitulated the drastic disruption of GJPs, the primary pathology of GJB2-related hearing loss. These in vitro models should be useful for establishing inner-ear cell therapies and drug screening that target GJB2-related hearing loss.

Novel allelic mutations in murine Serca2 induce differential development of squamous cell tumors.

Dominant mutations in the Serca2 gene, which encodes sarco(endo)plasmic reticulum calcium-ATPase, predispose mice to gastrointestinal epithelial carcinoma [1-4] and humans to Darier disease (DD) [14-17]. In this study, we generated mice harboring N-ethyl-N-nitrosourea (ENU)-induced allelic mutations in Serca2: three missense mutations and one nonsense mutation. Mice harboring these Serca2 mutations developed tumors that were categorized as either early onset squamous cell tumors (SCT), with development similar to null-type knockout mice [2,4] (aggressive form; M682, M814), or late onset tumors (mild form; M1049, M1162). Molecular analysis showed no aberration in Serca2 mRNA or protein expression levels in normal esophageal cells of any of the four mutant heterozygotes. There was no loss of heterozygosity at the Serca2 locus in the squamous cell carcinomas in any of the four lines. The effect of each mutation on Ca(2+)-ATPase activity was predicted using atomic-structure models and accumulated mutated protein studies, suggesting that putative complete loss of Serca2 enzymatic activity may lead to early tumor onset, whereas mutations in which Serca2 retains residual enzymatic activity result in late onset. We propose that impaired Serca2 gene product activity has a long-term effect on squamous cell carcinogenesis from onset to the final carcinoma stage through an as-yet unrecognized but common regulatory pathway.

Mouse models for ROS1-fusion-positive lung cancers and their application to the analysis of multikinase inhibitor efficiency.

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 early-onset 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 ROS1-fusion 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.

Identification of Reliable Components in Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS): a Data-Driven Approach across Metabolic Processes.

There is an increasing need to use multivariate statistical methods for understanding biological functions, identifying the mechanisms of diseases, and exploring biomarkers. In addition to classical analyses such as hierarchical cluster analysis, principal component analysis, and partial least squares discriminant analysis, various multivariate strategies, including independent component analysis, non-negative matrix factorization, and multivariate curve resolution, have recently been proposed. However, determining the number of components is problematic. Despite the proposal of several different methods, no satisfactory approach has yet been reported. To resolve this problem, we implemented a new idea: classifying a component as "reliable" or "unreliable" based on the reproducibility of its appearance, regardless of the number of components in the calculation. Using the clustering method for classification, we applied this idea to multivariate curve resolution-alternating least squares (MCR-ALS). Comparisons between conventional and modified methods applied to proton nuclear magnetic resonance ((1)H-NMR) spectral datasets derived from known standard mixtures and biological mixtures (urine and feces of mice) revealed that more plausible results are obtained by the modified method. In particular, clusters containing little information were detected with reliability. This strategy, named "cluster-aided MCR-ALS," will facilitate the attainment of more reliable results in the metabolomics datasets.

Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice.

BACKGROUND: Mutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness. The degeneration of the organ of Corti observed in Cx26 mutant-associated deafness is thought to be a secondary pathology of hearing loss. Here we focused on abnormal development of the organ of Corti followed by degeneration including outer hair cell (OHC) loss. METHODS: We investigated the crucial factors involved in late-onset degeneration and loss of OHC by ultrastructural observation, immunohistochemistry and protein analysis in our Cx26-deficient mice (Cx26f/fP0Cre). RESULTS: In ultrastructural observations of Cx26f/fP0Cre mice, OHCs changed shape irregularly, and several folds or notches were observed in the plasma membrane. Furthermore, the mutant OHCs had a flat surface compared with the characteristic wavy surface structure of OHCs of normal mice. Protein analysis revealed an increased protein level of caveolin-2 (CAV2) in Cx26f/fP0Cre mouse cochlea. In immunohistochemistry, a remarkable accumulation of CAV2 was observed in Cx26f/fP0Cre mice. In particular, this accumulation of CAV2 was mainly observed around OHCs, and furthermore this accumulation was observed around the shrunken site of OHCs with an abnormal hourglass-like shape. CONCLUSIONS: The deformation of OHCs and the accumulation of CAV2 in the organ of Corti may play a crucial role in the progression of, or secondary OHC loss in, GJB2-associated deafness. Investigation of these molecular pathways, including those involving CAV2, may contribute to the elucidation of a new pathogenic mechanism of GJB2-associated deafness and identify effective targets for new therapies.

Perinatal Gjb2 gene transfer rescues hearing in a mouse model of hereditary deafness.

Hearing loss is the most widespread sensory disorder, with an incidence of congenital genetic deafness of 1 in 1600 children. For many ethnic populations, the most prevalent form of genetic deafness is caused by recessive mutations in the gene gap junction protein, beta 2, 26 kDa (GJB2), which is also known as connexin 26 (Cx26). Despite this knowledge, existing treatment strategies do not completely recover speech perception. Here we used a gene delivery system to rescue hearing in a mouse model of Gjb2 deletion. Mice lacking Cx26 are characterized by profound deafness from birth and improper development of cochlear cells. Cochlear delivery of Gjb2 using an adeno-associated virus significantly improved the auditory responses and development of the cochlear structure. Using gene replacement to restore hearing in a new mouse model of Gjb2-related deafness may lead to the development of therapies for human hereditary deafness.

Deficiency of transcription factor Brn4 disrupts cochlear gap junction plaques in a model of DFN3 non-syndromic deafness.

Brn4, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome-linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K+ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.

Novel retinoblastoma mutation abrogating the interaction to E2F2/3, but not E2F1, led to selective suppression of thyroid tumors.

Mutant mouse models are indispensable tools for clarifying gene functions and elucidating the pathogenic mechanisms of human diseases. Here, we describe novel cancer models bearing point mutations in the retinoblastoma gene (Rb1) generated by N-ethyl-N-nitrosourea mutagenesis. Two mutations in splice sites reduced Rb1 expression and led to a tumor spectrum and incidence similar to those observed in the conventional Rb1 knockout mice. The missense mutant, Rb1(D326V/+) , developed pituitary tumors, but thyroid tumors were completely suppressed. Immunohistochemical analyses of thyroid tissue revealed that E2F1, but not E2F2/3, was selectively inactivated, indicating that the mutant Rb protein (pRb) suppressed thyroid tumors by inactivating E2F1. Interestingly, Rb1(D326V/+) mice developed pituitary tumors that originated from the intermediate lobe of the pituitary, despite selective inactivation of E2F1. Furthermore, in the anterior lobe of the pituitary, other E2F were also inactivated. These observations show that pRb mediates the inactivation of E2F function and its contribution to tumorigenesis is highly dependent on the cell type. Last, by using a reconstitution assay of synthesized proteins, we showed that the D326V missense pRb bound to E2F1 but failed to interact with E2F2/3. These results reveal the effect of the pRb N-terminal domain on E2F function and the impact of the protein on tumorigenesis. Thus, this mutant mouse model can be used to investigate human Rb family-bearing mutations at the N-terminal region.

Assembly of the cochlear gap junction macromolecular complex requires connexin 26.

Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

Autoimmune disorders associated with gain of function of the intracellular sensor MDA5.

MDA5 is an essential intracellular sensor for several viruses, including picornaviruses, and elicits antiviral interferon (IFN) responses by recognizing viral dsRNAs. MDA5 has been implicated in autoimmunity. However, the mechanisms of how MDA5 contributes to autoimmunity remain unclear. Here we provide direct evidence that dysregulation of MDA5 caused autoimmune disorders. We established a mutant mouse line bearing MDA5 mutation by ENU mutagenesis, which spontaneously developed lupus-like autoimmune symptoms without viral infection. Inflammation was dependent on an adaptor molecule, MAVS indicating the importance of MDA5-signaling. In addition, intercrossing the mutant mice with type I IFN receptor-deficient mice ameliorated clinical manifestations. This MDA5 mutant could activate signaling in the absence of its ligand but was paradoxically defective for ligand- and virus-induced signaling, suggesting that the mutation induces a conformational change in MDA5. These findings provide insight into the association between disorders of the innate immune system and autoimmunity.

Novel mouse model for Gardner syndrome generated by a large-scale N-ethyl-N-nitrosourea mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and elucidating the underlying pathogenic mechanisms of human diseases. We carried out large-scale mutagenesis using the chemical mutagen N-ethyl-N-nitrosourea. One specific aim of our mutagenesis project was to generate novel cancer models. We screened 7012 animals for dominant traits using a necropsy test and thereby established 17 mutant lines predisposed to cancer. Here, we report on a novel cancer model line that developed osteoma, trichogenic tumor, and breast cancer. Using fine mapping and genomic sequencing, we identified a point mutation in the adenomatous polyposis coli (Apc) gene. The Apc1576 mutants bear a nonsense mutation at codon 1576 in the Apc gene. Although most Apc mutant mice established thus far have multifocal intestinal tumors, mice that are heterozygous for the Apc1576 mutation do not develop intestinal tumors; instead, they develop multifocal breast cancers and trichogenic tumors. Notably, the osteomas that develop in the Apc1576 mutant mice recapitulate the lesion observed in Gardner syndrome, a clinical variant of familial adenomatous polyposis. Our Apc1576 mutant mice will be valuable not only for understanding the function of the Apc gene in detail but also as models of human Gardner syndrome.

SDOP-DB: a comparative standardized-protocol database for mouse phenotypic analyses.

UNLABELLED: This article reports the development of SDOP-DB, which can provide definite, detailed and easy comparison of experimental protocols used in mouse phenotypic analyses among institutes or laboratories. Because SDOP-DB is fully compliant with international standards, it can act as a practical foundation for international sharing and integration of mouse phenotypic information. AVAILABILITY: SDOP-DB (http://www.brc.riken.jp/lab/bpmp/SDOP/).

Introduction to the Japan Mouse Clinic at the RIKEN BioResource Center.

A systematic and comprehensive phenotyping platform has been developed by the RIKEN ENU-mutagenesis project between 1999 and 2007. As a result of phenotype screening on this platform, we have discovered about 400 mutants as animal models for human diseases. All information regarding these mouse mutants is now available to the public through our home page (http://www.brc.riken.jp/lab/gsc/mouse/indexJ.html). In 2008, we reconstructed the existing phenotyping platform and built a new platform. The new system has a hierarchical structure, consisting of a fundamental pipeline that utilizes the existing platform and an additional pipeline, which is optimized for more in-depth phenotyping assays. Using this system, we have started to perform more comprehensive phenotyping of mouse mutants. We have opened this system to Japanese scientists as the Japanese Mouse Clinic. It is anticipated that existing mouse mutants will be reevaluated as disease models by identifying novel phenotypes on the new platform. We will share detailed information about the standard operating procedures (SOPs) of our phenotyping analyses with other related large-scale projects, such as the European Mouse Disease Clinic (EUMODIC) and the German Mouse Clinic (GMC). Moreover, we will contribute to international efforts to standardize mouse phenotype data by sharing annotation of mutant phenotypes, which are made by internationally standardized methods, with other related projects.

Osteoporotic bone formation in mice lacking tob2; involvement of Tob2 in RANK ligand expression and osteoclasts differentiation.

Mice lacking tob2, a member of the antiproliferative family genes, had decreased bone mass, and the number of osteoclasts differentiated from bone marrow cells was increased. Overexpression of Tob2 in stromal cells repressed vitamin D(3)-induced osteoclasts formation. Furthermore, expression of RANKL mRNA in stromal cells was increased in the absence of Tob2 and decreased in the presence of Tob2. Tob2 interacted with vitamin D(3) receptor (VDR), which suggests its involvement in vitamin D(3) receptor-mediated regulation of transcription. Because VDR regulates RANKL expression, our data suggest that Tob2 negatively regulates formation of osteoclasts by suppressing RANKL expression through its interaction with VDR.

Carcinogenicity of dimethylarsinic acid in Ogg1-deficient mice.

Oxidative stress to DNA is recognized as a mechanism underlying carcinogenic effects of some environmental agents. Here, we hypothesized that dimethylarsinic acid (DMA(V)), an organic metabolite of inorganic arsenic in humans, might exert carcinogenic potential in a mouse line carrying a mutant Mmh allele of the Mmh/OGG1 gene encoding the enzyme 8-hydroxyguanine DNA glycosylase 1 (OGG1). Ogg1 mutant and wild type mice were treated with DMA(V) in their drinking water at a dose of 200 p.p.m. for up to 72 weeks. All DMA(V)-treated Ogg1(-/-)animals developed tumors, with a tendency for lower total incidences in the Ogg1(+/+) cases. Lung tumors in particular were induced as compared to the lack in non-carcinogen controls and were significantly more frequent in the homozygotes. At week 4, the levels of DNA 8-OH-dG and cell proliferation were significantly elevated in the lungs of non-treated Ogg1(-/-) as compared to Ogg1(+/+) mice and were strongly enhanced by DMA(V) treatment. Marked induction of Pola1, Cyp7b1, Ndfua3, Mmp13 and other genes specific to cell proliferation, cell signaling and xenobiotic metabolism in the lungs of DMA(V)-treated Ogg1(-/-) mice was found. Electron microscopic examination revealed the growth of microvilli, with increased numbers of mitochondria only in lungs and lung tumors of DMA(V)-exposed Ogg1(-/-) mice. Therefore, we strongly suggest that DMA(V) exerts carcinogenicity in the lungs of Ogg1(-/-) mutant mice, with a possible role for persistent accumulation of DNA oxidative adducts.

The study using wild-type and Ogg1 knockout mice exposed to potassium bromate shows no tumor induction despite an extensive accumulation of 8-hydroxyguanine in kidney DNA.

In order to assess the effect of potassium bromate (KBrO3) on the induction of tumor formation, a 1-year carcinogenesis study was performed using Ogg1 knockout mice (Ogg1(-/-)) and wild-type mice (Ogg1(+/+)). The mice were chronically exposed to KBrO3 by putting it in the drinking water for 29 weeks, at 2 g/l for the first 18 weeks, and then at 1 g/l for another 11 weeks. After termination of treatment the mice were kept for an additional 23 weeks. The amount of 8-hydroxydeoxyguanosine (8-OH-dG) in kidney DNA after 29 weeks of KBrO3 exposure reached 500 8-OH-dG/10(6) dG, almost 250-fold that of untreated wild-type mice. During the course of study the mice appeared normal, although a decrease of body weight gain in both Ogg1(-/-) and Ogg1(+/+) mice exposed to KBrO3, and some kidney malfunction in KBrO3 treated Ogg1(-/-) mice was observed. Surprisingly, when Ogg1(-/-) and Ogg1(+/+) mice were sacrificed at 52 weeks, no tumor formation could be found in kidney or other organs such as lung, liver, spleen, thymus, stomach and intestine. Microscopic examination also showed the absence of precancerous foci in all tissues of both Ogg1(-/-) and Ogg1(+/+) mice. A possible explanation is presented to reconcile these results with those of others which showed an increased incidence of tumor formation in untreated Ogg1(-/-) mice.

Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program.

SHIRPA is a three-stage protocol for the comprehensive assessment of primarily mouse behavior. The first stage consists of high-throughput phenotyping of 33 behavioral observations and 7 metabolic or disease observations. We modified this part of the protocol by integrating new morphologic observations into the initial phenotype assay of behavior and dysmorphology. Behavioral observations assessed by this protocol, now referred to as the "modified-SHIRPA," are compatible with the original "SHIRPA" protocol. Using modified-SHIRPA, we screened dominant phenotypes of more than 10,000 G(1) progeny generated by crossing DBA/2J females with ENU-treated C57BL/6J males. To date, we have obtained 136 hereditary-confirmed mutants that exhibit behavioral and morphologic defects. Some independent mutant lines exhibited similar phenotypes, suggesting that they may represent alleles of the same gene or mutations in the same genetic pathway. They could hold great potential for the unraveling of the molecular mechanisms of certain phenotypes.