Seiya Mizuno’s research while affiliated with University of Tsukuba and other places

BACKGROUND Aortic dissection (AD) is the separation of medial layers of the aorta and is a major cause of death in patients with connective tissue disorders such as Marfan syndrome. However, molecular triggers instigating AD, its temporospatial progression, and how vascular cells in each vessel layer interact and participate in the pathological process remain incompletely understood. To unravel the underlying molecular mechanisms of AD, we generated a spontaneous AD mouse model. METHODS We incorporated a novel missense variant (p.G234D) in FBN1 , the gene for fibrillin-1, identified in a patient with nonsyndromic familial AD into mice using the CRISPR/Cas9 system. We performed molecular pathological analyses of the aortic lesions by histology, immunofluorescence staining, electron microscopy, synchrotron-based imaging, and single-cell RNA sequencing. Biochemical analysis was performed to examine the binding capacity of mutant human FBN1G234D protein to LTBPs (latent TGFβ [transforming growth factor-beta] binding proteins), and signaling pathways in the mutant aortic wall were examined by the Western blot analysis. RESULTS Fifty percent of the Fbn1 G234D/G234D mutant mice died within 5 weeks of age from multiple intimomedial tears that expanded longitudinally and progressed to aortic rupture accompanied by massive immune cell infiltration. Fbn1 G234D/G234D endothelial cells exhibited altered mechanosensing with loss of parallel alignment to blood flow and upregulation of VCAM-1 and ICAM-1 as early as 1 week of age. Single-cell RNA sequencing, validated by immunostaining, revealed a cluster of monocyte/macrophage predominantly in the intima at 3 weeks of age before the dissection, and the second cluster of macrophages increased during the progression of intimomedial tears, exhibiting strong CCR2+ and both M1- and M2-like features. Consistently, upregulation of MMP2/9 was observed. Biochemically, FBN1G234D lost the ability to bind to LTBP-1, -2, and -4, resulting in the downregulation of TGFβ signaling in the aortic wall. CONCLUSIONS We show that interactions involving endothelial cells and macrophages/monocytes in the intima, where the ECM (extracellular matrix) microenvironment contains reduced TGFβ signaling, contribute to the initiation of AD. Our novel AD mouse model provides a unique opportunity to identify target molecules involved in the intimomedial tears that can be utilized for the development of therapeutic strategies.

Gastric mucosal homeostasis is maintained by tissue-resident stem and progenitor cells residing in the isthmus region. Following mucosal injury, surviving cells contribute to regeneration, coinciding with characteristic pathological changes such as atrophic gastritis and metaplasia. To comprehensively understand the cellular dynamics involved in this process, we performed single-cell and spatial transcriptomics using newly generated transgenic mice. In human samples and mouse models, loss of gastric chief cells precedes, and even induces, loss of parietal cells during the progression of atrophy and metaplasia, validating the causal relationship underlying the decrease of these two lineages. Single-cell analysis confirmed robust stemness and metaplastic changes in the Muc6 -expressing neck lineage following either chief or parietal cell ablation, and lineage-tracing experiments revealed that Muc6 -expressing isthmus progenitors serve as a source of metaplasia and regeneration. Mechanistically, mucosal injury recruits IL-1-expressing myeloid cells, which stimulates NRG1 production in stromal fibroblasts, leading to mucosal proliferation and regeneration mediated by Myc activation in isthmus progenitors. These findings highlight the injury-responsible stem cell-like function of Muc6 -expressing isthmal progenitors, which play a critical role in mucosal homeostasis and disease progression. Visual abstract

In Brief Tu et al. show that Tff2 ⁺ corpus isthmus cells are TA progenitors, and they, not chief cells, are the primary source of SPEM following injury. Upon Kras mutation, these progenitors directly progress to dysplasia, bypassing metaplasia, highlighting them as a potential origin of gastric cancer. Highlights Tff2 ⁺ corpus cells are TA progenitors that give rise to secretory cells. Tff2 ⁺ progenitors, not chief cells, are the primary source of SPEM after injury. Kras-mutant Tff2 ⁺ progenitors progress directly to dysplasia, bypassing metaplasia. Multi-omics analysis reveals distinct trajectories for SPEM and gastric cancer. Abstract Figure Graphical abstract Pyloric metaplasia, also known as spasmolytic polypeptide-expressing metaplasia (SPEM), arises in the corpus in response to oxyntic atrophy, but its origin and role in gastric cancer remain poorly understood. Using Tff2-CreERT knockin mice, we identified highly proliferative Tff2 ⁺ progenitors in the corpus isthmus that give rise to multiple secretory lineages, including chief cells. While lacking long-term self-renewal ability, Tff2 ⁺ corpus progenitors rapidly expand to form short-term SPEM following acute injury or loss of chief cells. Genetic ablation of Tff2 ⁺ progenitors abrogated SPEM formation, while genetic ablation of GIF ⁺ chief cells enhanced SPEM formation from Tff2 ⁺ progenitors. In response to H. pylori infection, Tff2 ⁺ progenitors progressed first to metaplasia and then later to dysplasia. Interestingly, induction of Kras G12D mutations in Tff2 ⁺ progenitors facilitated direct progression to dysplasia in part through the acquisition of stem cell-like properties. In contrast, Kras-mutated SPEM and chief cells were not able to progress to dysplasia. Tff2 mRNA was downregulated in isthmus cells during progression to dysplasia. Single-cell RNA sequencing and spatial transcriptomics of human tissues revealed distinct differentiation trajectories for SPEM and gastric cancer. These findings challenge the conventional interpretation of the stepwise progression through metaplasia and instead identify Tff2 ⁺ progenitor cells as potential cells of origin for SPEM and possibly for gastric cancer.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation and abnormal osteoclast activation, leading to bone destruction. We previously demonstrated that the large extracellular loop (LEL) of Tm4sf19 is important for its function in osteoclast differentiation, and LEL-Fc, a competitive inhibitor of Tm4sf19, effectively suppresses osteoclast multinucleation and prevent bone loss associated with osteoporosis. This study aimed to investigate the role of Tm4sf19 in RA, an inflammatory and abnormal osteoclast disease, using a mouse model of collagen-induced arthritis (CIA). Tm4sf19 expression was observed in macrophages and osteoclasts within the inflamed synovium, and Tm4sf19 expression was increased together with inflammatory genes in the joint bones of CIA-induced mice compared with the sham control group. Inhibition of Tm4sf19 by LEL-Fc demonstrated both preventive and therapeutic effects in a CIA mouse model, reducing the CIA score, swelling, inflammation, cartilage damage, and bone damage. Knockout of Tm4sf19 gene or inhibition of Tm4sf19 activity by LEL-Fc suppressed LPS/IFN-γ-induced TLR4-mediated inflammatory signaling in macrophages. LEL-Fc disrupted not only the interaction between Tm4sf19 and TLR4/MD2, but also the interaction between TLR4 and MD2. μCT analysis showed that LEL-Fc treatment significantly reduced joint bone destruction and bone loss caused by hyperactivated osteoclasts in CIA mice. Taken together, these findings suggest that LEL-Fc may be a potential treatment for RA and RA-induced osteoporosis by simultaneously targeting joint inflammation and bone destruction caused by abnormal osteoclast activation.

The creation of organisms with Cre-loxP conditional gene recombination systems often faces challenges, particularly when creating the initial (F0) generation with both a Cre recombinase and a DNA site flanked by loxP elements (floxed site). The primary reason is that it is difficult to synthesize a single plasmid with both the Cre gene and the floxed site, since Cre-mediated recombination spontaneously occurs when the plasmid is amplified in Escherichia coli bacterial cells. Here, we introduce an artificial nucleic acid sequence TATATATATATATATATA, named TAx9, that enables the integration of both the Cre gene and the floxed site into a single plasmid. TAx9 effectively blocks spontaneous Cre-mediated recombination in E. coli cells. Using this system, we created an F0 generation of transgenic newts and CRISPR-Cas9 knock-in mice with tissue-specific Cre recombination triggered by tamoxifen. TAx9 technology will be a powerful strategy for creating organisms capable of conditional genetic modification in the F0 generation, accelerating various life science research by reducing the time and cost for ultimately establishing and maintaining lines of genetically modified organisms.

Mast cells (MCs) play a central role in allergic immune responses. MC activation is regulated by several inhibitory immunoreceptors. The CD300 family members CD300a and CD300lf recognize phospholipid ligands and inhibit the FcεRI-mediated activating signal in MCs. While CD300a binds to phosphatidylserine (PS) to inhibit MCs activation, CD300lf function is less clear due to its ability to bind with ceramide and PS. Moreover, it also remains blurring whether CD300a and CD300lf function independently, cooperatively, or by interfering with each other in regulating MC activation. Using imaging and flow cytometric analyses of bone marrow-derived cultured MCs (BMMCs) from wild-type (WT), Cd300a–/–, Cd300lf–/–, and Cd300a–/–Cd300lf–/– mice, we show that CD300lf and CD300a colocalized with PS externalized to the outer leaflet of the plasma membrane with a polar formation upon activation, and CD300lf cooperates with CD300a to inhibit BMMCs activation. CD300lf also colocalized with extracellular ceramide in addition to the internal PS on the cell surface, which results in stronger inhibition of MC activation than CD300lf binding to PS alone. Similarly, although both Cd300a–/– and Cd300lf–/– mice showed decreased rectal temperatures compared with WT mice in the model of passive systemic anaphylaxis, Cd300a–/–Cd300lf–/– mice showed lower rectal temperature than either Cd300a–/– or Cd300lf–/– mice. Our results demonstrate the cooperativity of multiple inhibitory receptors expressed on MCs and their regulatory functions upon binding to respective ligands.

In vivo cell type‐specific genetic recombination based on the Cre‐loxP system has contributed to the understanding of biological processes and diseases. Neuronal nuclei (NeuN)/RBFOX3 is a widely used mature neuron marker in developmental biology and neuroscience. Here, we generated Rbfox3‐improved Cre (iCre) knock‐in mouse model and investigated the effect of iCre knock‐in into the Rbfox3 gene and Cre recombination activity in the central nervous system (CNS) and peripheral tissues. The knock‐in of internal ribosome entry site (IRES)‐iCre cassette into the Rbfox3 3′ UTR did not affect birth rate, growth, and brain weight. In the adult brain, iCre protein expression was confirmed, whereas RBFOX3 protein expression was partially reduced in the knock‐in mice. Cre recombination analysis using R26GRR fluorescent reporter strain revealed that Rbfox3‐driven iCre‐induced gene recombination in the CNS and heart during embryonic development. In the adult brain, gene recombination was observed in neurons, however, not in other glial cells. In the peripheral tissues, iCre activity was found in the sciatic nerve and in other peripheral tissues, including the heart, bladder, and testis. We validated gene recombination rate in the germline and found that 100% recombination occurred in male germ cells and approximately 50% in female germ cells. Concludingly, Rbfox3‐iCre mice induce genetic recombination in neurons within CNS as well as in some peripheral tissues and germ cells. In addition to establishing a novel Cre mouse line, the findings of this study offer valuable insights into the development and application of mouse tools that utilize the Rbfox3 gene locus.

A limited number of female germ cells support reproduction in many mammals. The follicle, composed of oocytes and supporting granulosa cells, forms the basis of oogenesis. Crosstalk between oocytes and granulosa cells is essential for the formation, dormancy, re-awakening, and maturation of oocytes. The oocyte expresses c-KIT and growth differentiation factor-9 (GDF-9), which are major factors in this crosstalk. The downstream signalling pathways of c-KIT and GDF-9 have been well-documented; however, their intra-oocyte trafficking pathway remains unclear. Our study reveals that the exocyst complex, a heterotetrameric protein complex important for tethering in vesicular transport, is important for proper intra-oocyte trafficking of c-KIT and GDF9 in mice. We found that depletion of oocyte-specific EXOC1, a component of the exocyst complex, impaired oocyte re-awakening and cyst breakdown, and inhibited granulosa cell proliferation during follicle growth. The c-KIT receptor is localised on the oocyte plasma membrane. The oocyte-specific Kit conditional knockout mice were reported to exhibit impaired oocyte re-awakening and reduced oocyte cyst breakdown. GDF9 is a protein secreted extracellularly in the oocyte. Previous studies have shown that Gdf9 knockout mice impaired proliferation and granulosa cell multilayering in growing follicles. We found that both c-KIT and GDF9 abnormally stuck in the EXOC1-depleted oocyte cytoplasm. These abnormal phenotypes were also observed in oocytes depleted of exocyst complex members EXOC3 and EXOC7. These results clearly show that the exocyst complex is essential for proper intra-oocyte trafficking of c-KIT and GDF9. Inhibition of this complex causes complete loss of female fertility in mice. Our findings build a platform for research related to trafficking mechanisms of vital crosstalk factors for oogenesis.

With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, increasing the risk of chromosomal rearrangements and posing a major obstacle to simultaneous multiple gene knockout. Base-editing systems, such as Target-AID, are safe alternatives for precise base modifications without requiring DNA double-strand breaks, serving as promising solutions for existing challenges. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockout highlights the need for a comprehensive system to design guide RNAs (gRNAs) for the simultaneous knockout of multiple genes. Here, we aimed to develop KOnezumi-AID, a command-line tool for gRNA design for Target-AID-mediated genome editing. KOnezumi-AID facilitates gene knockout by inducing the premature termination codons or promoting exon skipping, thereby generating experiment-ready gRNA designs for mouse and human genomes. Additionally, KOnezumi-AID exhibits batch processing capacity, enabling rapid and precise gRNA design for large-scale genome editing, including CRISPR screening. In summary, KOnezumi-AID is an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.

Study Objectives Sleep/wakefulness is regulated by intracellular signaling pathways composed of protein kinases such as salt-inducible kinase 3 (Sik3). Sik3-deficiency in neurons decreases nonrapid eye movement (NREM) sleep time and electroencephalogram (EEG) delta power during NREM sleep, while Sik3Slp mice lacking a protein kinase A (PKA)-phosphorylation site, S551, show hypersomnia phenotype. In this study, we examined how a phosphomimetic mutation of the 221st threonine residue (T221E), which provides a partial (weak) constitutive activity of the kinase, affects sleep/wakefulness and circadian behavior. We also examined the effect of T221E substitution on the hypersomnia phenotype of Sik3Slp mice. Methods We examined the sleep/wake behavior of heterozygous and homozygous Sik3T221E mice and Sik3T221E;Slp mice using EEG and electromyogram recording. We also examined the circadian behavior of Sik3T221E mice using a running wheel under the light–dark cycle and constant darkness. Results Heterozygous and homozygous Sik3T221E mice showed normal sleep time and sleep homeostatic responses. Homozygous Sik3T221E mice exhibited a delayed onset of wakefulness at the early dark phase and longer circadian periods. Sik3T221E;Slp mice showed decreased NREM sleep time and homeostatic responses compared to Sik3Slp mice. Conclusions Our results suggest that the peak onset of wakefulness is sensitive to disturbed kinase activity of SIK3, and the relationship between phosphorylation at T221 and S551 is critical for regulating sleep need.