Kazuhiro Okumura

The complete mitochondrial genome of the domestic red deer (Cervus elaphus) of New Zealand and its phylogenic position within the family Cervidae.

We determined the complete nucleotide sequence of the mitochondrial genome of the semidomestic red deer (Cervus elaphus) of New Zealand. The genome was 16,357 bp long and contained 13 protein-coding genes, 12SrRNA, 16SrRNA, 22 tRNAs and a D-loop as found in other mammals. Database homology searches showed that the mitochondrial DNA (mtDNA) sequence from the New Zealand semidomestic deer was similar to partial mtDNA sequences from the European, Norwegian (C. e. atlanticus) and Spanish red deer (C. e. hispanicus). Phylogenetic analysis of the mitochondrial protein-coding regions revealed two well-defined monophyletic clades in subfamilies Cervinae and Muntiacinae. However, red deer and Sika deer were not found to be close relatives. The analysis did identify the red deer as a sister taxon of a Samber/Sika deer clade, although it was more closely related to the Samber than the Sika group.

Meis1 Regulates Epidermal Stem Cells and Is Required for Skin Tumorigenesis

Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1.

Protein 4.1 expression in the developing hair cells of the mouse inner ear.

Protein 4.1 (band 4.1 or 4.1R) was originally identified as an abundant protein of the human erythrocyte, in which it stabilizes the spectrin/actin cytoskeleton. Subsequently, several new family members, 4.1N, 4.1G and 4.1B, have been identified, which are expressed in many cell types, in particular at cell-cell junctions. We previously reported that 4.1R and 4.1N are expressed in the inner ear hair cells with specific localization patterns, and that 4.1R forms a complex with the membrane-associated guanylate kinase (MAGUK) protein p55 and two deafness gene products, myosin XV and whirlin. To determine the functions of the other family members, 4.1G and 4.1B, we observed their expression patterns in developing stereocilia in mice inner ear hair cells. 4.1G is expressed in the basal tapers of the stereocilia bundle in early postnatal stages. 4.1B was specifically and constantly expressed in the stereocilia tips during postnatal development. Additionally, we found that 4.1B is ablated in the hair cells of both myosin XV and whirlin mutant mice at all stages in hair cell development. These results suggest that 4.1 family members play important roles in the development and maintenance of the inner ear hair cells, and that 4.1B may be a member of the myosin XV-whirlin complex that is important for stereocilia maturation.

Independent genetic control of early and late stages of chemically induced skin tumors in a cross of a Japanese wild-derived inbred mouse strain, MSM/Ms

MSM/Ms is an inbred mouse strain derived from a Japanese wild mouse, Mus musculus molossinus. In this study, we showed that MSM/Ms mice exhibit dominant resistance when crossed with susceptible FVB/N mice and subjected to the two-stage skin carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA)/ 12-O-tetradecanoylphorbol-13-acetate (TPA). A series of F1 backcross mice were generated by crossing p53(+/+) or p53(+/-) F1 (FVB/N × MSM/Ms) males with FVB/N female mice. These generated 228 backcross animals, approximately half of which were p53(+/-), enabling us to search for p53-dependent skin tumor modifier genes. Highly significant linkage for papilloma multiplicity was found on chromosomes 6 and 7 and suggestive linkage was found on chromosomes 3, 5 and 12. Furthermore, in order to identify stage-dependent linkage loci we classified tumors into three categories (<2mm, 2-6mm and >6mm), and did linkage analysis. The same locus on chromosome 7 showed strong linkage in groups with <2mm or 2-6mm papillomas. No linkage was detected on chromosome 7 to papillomas >6mm, but a different locus on chromosome 4 showed strong linkage both to papillomas >6mm and to carcinomas. This locus, which maps near the Cdkn2a/p19(Arf) gene, was entirely p53-dependent, and was not seen in p53 (+/-) backcross animals. Suggestive linkage conferring susceptibility to carcinoma was also found on chromosome 5. These results clearly suggest distinct loci regulate each stage of tumorigenesis, some of which are p53-dependent.

Quantitative trait loci on chromosome 5 for susceptibility to frequency-specific effects on hearing in DBA/2J mice

The DBA/2J strain is a model for early-onset, progressive hearing loss in humans, as confirmed in the present study. DBA/2J mice showed progression of hearing loss to low-frequency sounds from ultrasonic-frequency sounds and profound hearing loss at all frequencies before 7 months of age. It is known that the early-onset hearing loss of DBA/2J mice is caused by affects in the ahl (Cdh23ahl) and ahl8 (Fscn2ahl8) alleles of the cadherin 23 and fascin 2 genes, respectively. Although the strong contributions of the Fscn2ahl8 allele were detected in hearing loss at 8- and 16-kHz stimuli with LOD scores of 5.02 at 8 kHz and 8.84 at 16 kHz, hearing loss effects were also demonstrated for three new quantitative trait loci (QTLs) for the intervals of 50.3–54.5, 64.6–119.9, and 119.9–137.0 Mb, respectively, on chromosome 5, with significant LOD scores of 2.80–3.91 for specific high-frequency hearing loss at 16 kHz by quantitative trait loci linkage mapping using a (DBA/2J × C57BL/6J) F1 × DBA/2J backcross mice. Moreover, we showed that the contribution of Fscn2ahl8 to early-onset hearing loss with 32-kHz stimuli is extremely low and raised the possibility of effects from the Cdh23ahl allele and another dominant quantitative trait locus (loci) for hearing loss at this ultrasonic frequency. Therefore, our results suggested that frequency-specific QTLs control early-onset hearing loss in DBA/2J mice.

Congenic Mapping and Allele-Specific Alteration Analysis of Stmm1 Locus Conferring Resistance to Early-Stage Chemically Induced Skin Papillomas

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.

Identification of Stmm3 locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F1 × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N2 and N10 congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.

The parathyroid hormone regulates skin tumour susceptibility in mice

Using a forward genetics approach to map loci in a mouse skin cancer model, we previously identified a genetic locus, Skin tumour modifier of MSM 1 (Stmm1) on chromosome 7, conferring strong tumour resistance. Sub-congenic mapping localized Parathyroid hormone (Pth) in Stmm1b. Here, we report that serum intact-PTH (iPTH) and a genetic polymorphism in Pth are important for skin tumour resistance. We identified higher iPTH levels in sera from cancer-resistant MSM/Ms mice compared with susceptible FVB/NJ mice. Therefore, we performed skin carcinogenesis experiments with MSM-BAC transgenic mice (PthMSM-Tg) and Pth knockout heterozygous mice (Pth+/−). As a result, the higher amounts of iPTH in sera conferred stronger resistance to skin tumours. Furthermore, we found that the coding SNP (rs51104087, Val28Met) localizes in the mouse Pro-PTH encoding region, which is linked to processing efficacy and increased PTH secretion. Finally, we report that PTH increases intracellular calcium in keratinocytes and promotes their terminal differentiation. Taken together, our data suggest that Pth is one of the genes responsible for Stmm1, and serum iPTH could serve as a prevention marker of skin cancer and a target for new therapies.

DEclust: A statistical approach for obtaining differential expression profiles of multiple conditions

High-throughput RNA sequencing technology is widely used to comprehensively detect and quantify cellular gene expression. Thus, numerous analytical methods have been proposed for identifying differentially expressed genes (DEGs) between paired samples such as tumor and control specimens, but few studies have reported methods for analyzing differential expression under multiple conditions. We propose a novel method, DEclust, for differential expression analysis among more than two matched samples from distinct tissues or conditions. As compared to conventional clustering methods, DEclust more accurately extracts statistically significant gene clusters from multi-conditional transcriptome data, particularly when replicates of quantitative experiments are available. DEclust can be used for any multi-conditional transcriptome data, as well as for extending any DEG detection tool for paired samples to multiple samples. Accordingly, DEclust can be used for a wide range of applications for transcriptome data analysis. DEclust is freely available at http://www.dna.bio.keio.ac.jp/software/DEclust.

Time-Series Analysis of Tumorigenesis in a Murine Skin Carcinogenesis Model

Recent years have witnessed substantial progress in understanding tumor heterogeneity and the process of tumor progression; however, the entire process of the transition of tumors from a benign to metastatic state remains poorly understood. In the present study, we performed a prospective cancer genome-sequencing analysis by employing an experimental carcinogenesis mouse model of squamous cell carcinoma to systematically understand the evolutionary process of tumors. We surgically collected a part of a lesion of each tumor and followed the progression of these tumors in vivo over time. Comparative time-series analysis of the genomes of tumors with different fates, i.e., those that eventually metastasized and regressed, suggested that these tumors acquired and inherited different mutations. These findings suggest that despite the occurrence of an intra-tumor selection event for malignant alteration during the transformation from early- to late-stage papilloma, the fate determination of tumors might be determined at an even earlier stage.