Publications by authors named "Shinya Ayabe"

15 Publications

  • Page 1 of 1

Engrailed Homeobox 1 and Cytokeratin 19 Are Independent Diagnostic Markers of Eccrine Porocarcinoma and Distinguish It From Squamous Cell Carcinoma.

Am J Clin Pathol 2020 09;154(4):499-509

Department of Human Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

Objectives: The diagnostic utility of En1 in the histopathologic differentiation of eccrine porocarcinoma (EPC) from invasive squamous cell carcinoma (SCC) was investigated.

Methods: Expression of En1 and CK19 in 16 cases of EPC was immunohistochemically examined and compared with that in 32 cases of SCC.

Results: In all 16 EPCs, En1 was expressed in 3% to 100% of tumor cells. In 20 of the 32 SCCs, En1 was expressed in 3% to 90% of tumor cells. A total of 13 of the 16 EPCs and five of the 32 SCCs were judged as En1 positive, with a cutoff value of 25%. In addition, 11 of the 16 EPCs and four of the 32 SCCs were CK19 positive. The frequencies of En1- and CK19-positive cases were significantly higher in EPCs than in SCCs. In a logistic regression analysis for predicting EPC, En1 and CK19 were independent markers. When expression patterns of En1 and CK19 were combined, none of the 32 SCCs was both positive. In contrast, 15 of the 16 EPCs were positive for either En1 or CK19.

Conclusions: A combination of En1 and CK19 expression can improve the accuracy of histologic diagnosis of EPC.
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http://dx.doi.org/10.1093/ajcp/aqaa066DOI Listing
September 2020

Reverse genetics reveals single gene of every candidate on Hybrid sterility, X Chromosome QTL 2 (Hstx2) are dispensable for spermatogenesis.

Sci Rep 2020 06 3;10(1):9060. Epub 2020 Jun 3.

Laboratory Animal Resource Center and Trans-border Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan.

F1 hybrid progenies between related subspecies often show hybrid sterility (HS) or inviability. HS is caused by failure of meiotic chromosome synapsis and sex body formation in house mouse. Previous studies identified two HS critical genomic regions named Hstx2 on Chr X and Hst1 on Chr 17 by murine forward genetic approaches. HS gene on Hst1 was reported to be Prdm9. Intersubspecific polymorphisms of Prdm9 induce HS in hybrids, and Prdm9 null mutation leads to sterility in the inbred strain. However, HS gene on Hstx2 remains unknown. Here, using knock-out studies, we showed that HS candidate genes on Hstx2 are not individually essential for spermatogenesis in B6 strain. We examined 12 genes on Hstx2: Ctag2, 4930447F04Rik, Mir743, Mir465d, Mir465c-2, Mir465b-1, Mir465c-1, Mir465, Gm1140, Gm14692, 4933436I01Rik, and Gm6812. These genes were expressed in adult testes, and showed intersubspecific polymorphisms on expressed regions. This first reverse genetic approach to identify HS gene on Hstx2 suggested that the loss of function of any one HS candidate gene does not cause complete sterility, unlike Prdm9. Thus, the mechanism(s) of HS by the HS gene on Hstx2 might be different from that of Prdm9.
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http://dx.doi.org/10.1038/s41598-020-65986-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270182PMC
June 2020

Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.

Methods 2021 07 22;191:23-31. Epub 2020 Apr 22.

Laborarory Animal Resource Center, Transborder Medical Research Center, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.
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http://dx.doi.org/10.1016/j.ymeth.2020.04.007DOI Listing
July 2021

Generation of B6-Ddx4 , a novel CreERT2 knock-in line, for germ cell lineage by CRISPR/Cas9.

Genesis 2020 07 15;58(7):e23367. Epub 2020 Apr 15.

Trans-Border Medical Research Center, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.

Germ cell development is essential for maintaining reproduction in animals. In postpubertal females, oogenesis is a highly complicated event for producing fertilizable oocytes. It starts when dormant primordial oocytes undergo activation to become growing oocytes. In postpubertal males, spermatogenesis is a differentiation process for producing sperm from spermatogonial stem cells. To obtain full understanding of the molecular mechanisms underlying germ cell development, the Cre/loxP system has been widely applied for conditional knock-out mouse studies. In this study, we established a novel knock-in mouse line, B6-Ddx4 , which expresses CreERT2 recombinase under the control of the endogenous DEAD-box helicase 4 (Ddx4) gene promoter. Ddx4 was specifically expressed in both female and male germ cell lineages. We mated the CreERT2 mice with R26GRR mice, expressing enhanced green fluorescent protein (EGFP) and tDsRed before and after Cre recombination. We found tDsRed signals in the testes and ovaries of tamoxifen-treated B6-Ddx4 ::R26GRR mice, but not in untreated mice. Immunostaining of their ovaries clearly showed that Cre recombination occurred in all oocytes at every follicle stage. We also found 100% Cre recombination efficiency in male germ cells via the progeny test. In summary, our results indicate that B6-Ddx4 is beneficial for studying female and male germ cell development.
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http://dx.doi.org/10.1002/dvg.23367DOI Listing
July 2020

Pathogenic POGZ mutation causes impaired cortical development and reversible autism-like phenotypes.

Nat Commun 2020 02 26;11(1):859. Epub 2020 Feb 26.

Technology and Developmental Team for Mouse Phenotype Analysis, RIKEN BioResource Research Center, Tsukuba, Ibaraki, 305-0074, Japan.

Pogo transposable element derived with ZNF domain (POGZ) has been identified as one of the most recurrently de novo mutated genes in patients with neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability and White-Sutton syndrome; however, the neurobiological basis behind these disorders remains unknown. Here, we show that POGZ regulates neuronal development and that ASD-related de novo mutations impair neuronal development in the developing mouse brain and induced pluripotent cell lines from an ASD patient. We also develop the first mouse model heterozygous for a de novo POGZ mutation identified in a patient with ASD, and we identify ASD-like abnormalities in the mice. Importantly, social deficits can be treated by compensatory inhibition of elevated cell excitability in the mice. Our results provide insight into how de novo mutations on high-confidence ASD genes lead to impaired mature cortical network function, which underlies the cellular pathogenesis of NDDs, including ASD.
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http://dx.doi.org/10.1038/s41467-020-14697-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044294PMC
February 2020

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

Genome Biol 2019 08 26;20(1):171. Epub 2019 Aug 26.

Department of Basic Medicine, Division of Basic Medical Science and Molecular Medicine, School of Medicine, Tokai University, 143, Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.

Background: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method).

Results: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach.

Conclusion: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.
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http://dx.doi.org/10.1186/s13059-019-1776-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709553PMC
August 2019

Off- and on-target effects of genome editing in mouse embryos.

J Reprod Dev 2019 Feb 6;65(1):1-5. Epub 2018 Dec 6.

Experimental Animal Division, RIKEN BioResource Research Center, Ibaraki 305-0074, Japan.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based genome editing technology has enabled manipulation of the embryonic genome. Unbiased whole genome sequencing comparing parents to progeny has revealed that the rate of Cas9-induced mutagenesis in mouse embryos is indistinguishable from the background rate of de novo mutation. However, establishing the best practice to confirm on-target alleles of interest remains a challenge. We believe that improvement in editing strategies and screening methods for founder mice will contribute to the generation of quality-controlled animals, thereby ensuring reproducibility of results in animal studies and advancing the 3Rs (replacement, reduction, and refinement).
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http://dx.doi.org/10.1262/jrd.2018-128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379761PMC
February 2019

A mutation in transcription factor MAFB causes Focal Segmental Glomerulosclerosis with Duane Retraction Syndrome.

Kidney Int 2018 08 18;94(2):396-407. Epub 2018 May 18.

Division of Nephrology, Department of Medicine, Showa University Fujigaoka Hospital, Fujigaoka, Japan.

Focal segmental glomerulosclerosis (FSGS) is a leading cause of end-stage renal disease in children and adults. Genetic factors significantly contribute to early-onset FSGS, but the etiologies of most adult cases remain unknown. Genetic studies of monogenic syndromic FSGS exhibiting extra-renal manifestations have uncovered an unexpected biological role for genes in the development of both podocytes and other cellular lineages. To help define these roles, we studied two unrelated families with FSGS associated with Duane Retraction Syndrome, characterized by impaired horizontal eye movement due to cranial nerve malformation. All four affected individuals developed FSGS and Duane Retraction Syndrome in their first to second decade of life, manifested as restricted abduction together with globe retraction and narrowed palpebral fissure on attempted adduction. Hypoplasia of the abducens nerves and hearing impairment occurred in severely affected individuals. Genetic analyses revealed that affected individuals harbor a rare heterozygous substitution (p.Leu239Pro) in MAFB, a leucine zipper transcription factor. Luciferase assays with cultured monocytes indicated that the substitution significantly reduced transactivation of the F4/80 promoter, the known MAFB recognition element. Additionally, immunohistochemistry indicated reduced MAFB expression in the podocytes of patients. Structural modeling suggested that the p.Leu239Pro substitution in the DNA-binding domain possibly interferes with the stability of the adjacent zinc finger. Lastly, podocytes in neonatal mice with p.Leu239Pro displayed impaired differentiation. Thus, MAFB mutations impair development and/or maintenance of podocytes, abducens neurons and the inner ear. The interactions between MAFB and regulatory elements in these developing organs are likely highly specific based on spatiotemporal requirements.
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http://dx.doi.org/10.1016/j.kint.2018.02.025DOI Listing
August 2018

Homeobox transcriptional factor engrailed homeobox 1 is expressed specifically in normal and neoplastic sweat gland cells.

Histopathology 2018 Jun 25;72(7):1199-1208. Epub 2018 Mar 25.

Department of Human Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.

Aims: A number of homeobox transcriptional factors are utilised as organ-specific markers in the histopathological diagnosis of neoplasms. We have screened a homeobox gene that is expressed specifically in normal sweat gland cells and is useful for the histopathological diagnosis of sweat gland neoplasms.

Methods And Results: By screening an open database resource of The Human Protein Atlas, 37 genes among the 235 homeobox transcriptional factors were found to be expressed specifically in the skin. Among those 37 genes, the engrailed homeobox 1 (En1) was expressed in normal eccrine glands but not in the epidermal keratinocytes. Expression of En1 was found throughout the eccrine glands, but not in the apocrine secretory coils, sebaceous glands or hair follicles. Expression of En1 was examined immunohistochemically in 111 cases of cutaneous epithelial neoplasms. All nine cases of poroma, seven cases of spiradenoma and six cases of syringoma, which are considered to differentiate towards eccrine glands, showed positive nuclear staining in most of the tumour cells. Sebaceous gland and hair follicle tumours were immunonegative. En1 was expressed focally in the epidermal neoplasms of seborrheic keratosis and squamous cell carcinoma.

Conclusion: Engrailed homeobox 1 was expressed specifically in normal eccrine glands and was expressed in most of the tumour cells of sweat gland neoplasms with eccrine gland differentiation. En1 was expressed focally in epidermal neoplasms; however, it was absent in sebaceous or hair follicle neoplasms. These findings will help in the histopathological diagnosis as well as understanding of the histogenesis of sweat gland neoplasms.
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http://dx.doi.org/10.1111/his.13486DOI Listing
June 2018

Prostaglandin D2 Attenuates Bleomycin-Induced Lung Inflammation and Pulmonary Fibrosis.

PLoS One 2016 19;11(12):e0167729. Epub 2016 Dec 19.

Department of Animal Radiology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan.

Pulmonary fibrosis is a progressive and fatal lung disease with limited therapeutic options. Although it is well known that lipid mediator prostaglandins are involved in the development of pulmonary fibrosis, the role of prostaglandin D2 (PGD2) remains unknown. Here, we investigated whether genetic disruption of hematopoietic PGD synthase (H-PGDS) affects the bleomycin-induced lung inflammation and pulmonary fibrosis in mouse. Compared with H-PGDS naïve (WT) mice, H-PGDS-deficient mice (H-PGDS-/-) represented increased collagen deposition in lungs 14 days after the bleomycin injection. The enhanced fibrotic response was accompanied by an increased mRNA expression of inflammatory mediators, including tumor necrosis factor-α, monocyte chemoattractant protein-1, and cyclooxygenase-2 on day 3. H-PGDS deficiency also increased vascular permeability on day 3 and infiltration of neutrophils and macrophages in lungs on day 3 and 7. Immunostaining showed that the neutrophils and macrophages expressed H-PGDS, and its mRNA expression was increased on day 3and 7 in WT lungs. These observations suggest that H-PGDS-derived PGD2 plays a protective role in bleomycin-induced lung inflammation and pulmonary fibrosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167729PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167321PMC
July 2017

Prostaglandin D2 inhibits collagen secretion from lung fibroblasts by activating the DP receptor.

J Pharmacol Sci 2013 29;121(4):312-7. Epub 2013 Mar 29.

Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Japan.

Lung fibroblasts are responsible for collagen secretion during normal tissue repair and the development of fibrosis. Many other prostaglandins have been reported to regulate collagen synthesis in lung fibroblasts, but the role of prostaglandin D2 (PGD2) is unknown. In this study, we investigated the effect of PGD2 on type I collagen secretion in human lung fibroblasts. Pretreatment with PGD2 (0.1 - 10 μM, 1 h) significantly attenuated type I collagen secretion to the cell supernatant induced by transforming growth factor-β (TGF-β). Although an agonist on chemoattractant receptorhomologous molecule expressed on Th2 cells (CRTH2) did not have any effect, the prostanoid DP-receptor agonist BW245C (0.01 - 1 μM) suppressed TGF-β-induced collagen secretion. PGD2 and BW245C significantly increased intracellular cAMP level. One-hour pretreatment with forskolin (0.1 - 10 μM), dibutyryl-cAMP (0.01 - 1 mM), and the protein kinase A (PKA)-activator N(6)-phenyl-cyclic AMP (100 μM) significantly reduced TGF-β-induced collagen secretion, while exchange protein activated by cAMP (Epac) activator 8-bromo-2'-O-methyladenosine-3',5'-cyclic AMP (10 μM) did not affect collagen deposition. These results suggest that PGD2 inhibits TGF-β-induced collagen secretion via intracellular cAMP accumulation through activating DP receptor.
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http://dx.doi.org/10.1254/jphs.12275fpDOI Listing
October 2013

Relaxant effect of prostaglandin D(2)--receptor DP agonist on liver myofibroblast contraction.

J Pharmacol Sci 2011 25;116(2):197-203. Epub 2011 May 25.

Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Japan.

Increased intrahepatic resistance causes portal hypertension in cirrhosis. Liver myofibroblasts (MFs) are now regarded as the principle cells involved in sinusoidal blood flow regulation. Many other prostaglandin-receptor agonists have been reported to regulate liver MF contraction, but the role of the prostaglandin D(2)-receptor DP is unknown. In this study, we investigated the effect of a synthetic agonist of prostanoid DP receptor, BW245C, on contractile properties of primary rat liver MFs. Collagen gel contraction assay revealed that BW245C alone (1 and 10 µM) did not induce contraction but induced cell relaxation. Pretreatment with BW245C (10 µM, 30 min) attenuated bradykinin (100 nM)-induced liver MF contraction. Elevation of [Ca(2+)](i) induced by bradykinin (100 nM) was partially suppressed by BW245C pretreatment (10 µM, 3 min). BW245C (1 and 10 µM) significantly increased intracellular cAMP level in a dose-dependent manner. Pretreatment with forskolin (30 - 300 nM, 30 min) and dibutyryl-cAMP (3 - 30 µM, 30 min) significantly reduced bradykinin-induced contraction. Furthermore, a protein kinase A (PKA) inhibitor KT5720 (10 nM to 1 µM, 30 min) blocked the relaxant effect of BW245C. These results suggest that prostanoid DP receptor agonism inhibits bradykinin-induced [Ca(2+)](i) elevation and contraction through cAMP-PKA signal activation in rat liver MFs.
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http://dx.doi.org/10.1254/jphs.10325fpDOI Listing
October 2011

Prostaglandin D(2) induces contraction via thromboxane A(2) receptor in rat liver myofibroblasts.

Eur J Pharmacol 2008 Sep 14;591(1-3):237-42. Epub 2008 Jun 14.

Department of Veterinary Pharmacology, Agriculture and Life Science, The University of Tokyo, Japan.

Increased intrahepatic resistance is one of the major characteristics of cirrhotic liver, in which extravascular cells including liver myofibroblasts (MFs) abnormally contract. Although several studies provided evidence that various prostaglandins (PG) are involved in liver cirrhosis, the role of PGD(2) remains unknown. In this study, we investigated the effect of PGD(2) on the contractile properties of liver MFs. Cultured rat liver MFs were used at passages 4-7. A collagen gel contraction assay was used for the evaluation of the MFs contraction. mRNA expression was assessed by semi-quantitative RT-PCR. Intracellular Ca(2+) concentrations ([Ca(2+)](i)) were measured by monitoring the fluorescence intensity of fura-2. PGD(2) (1-10 microM) induced liver MF contraction in a dose-dependent manner with [Ca(2+)](i) elevation. Pretreatment with 300 nM LaCl(3), a nonselective Ca(2+) channel blocker abolished the 10 microM PGD(2)-induced MFs contraction. RT-PCR revealed that three distinct PGD(2) responsive receptors, prostanoid DP receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) and thromboxane A(2) receptor (prostanoid TP receptor), were expressed in liver MFs. While prostanoid DP receptor agonist and CRTH2 agonist didn't induce contraction, 0.01-1 microM U46619 (11alpha, 9alpha-epoxymethano-PGH(2), prostanoid TP receptor agonist) caused robust contraction with [Ca(2+)](i) elevation. Furthermore, pretreatment with prostanoid TP receptor antagonists ramatroban (1 microM) or SQ29548 ([1S-[1alpha, 2alpha(Z), 3alpha, 4alpha]]-7-[3-[[2-[(phenyl amino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid, 1 microM) completely suppressed PGD(2)-induced contraction and [Ca(2+)](i) elevation. Additionally, we observed that BW245C (1-10 microM) decreased basal MF contraction. These results suggest that PGD(2) induces rat liver MF contraction with an increase in [Ca(2+)](i) through prostanoid TP receptor.
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http://dx.doi.org/10.1016/j.ejphar.2008.06.037DOI Listing
September 2008