Immunoglobulin A-specific deficiency induces spontaneous inflammation specifically in the ileum.

OBJECTIVE: Although immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism. DESIGN: We generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging. RESULTS: We obtained two lines, with one that contained a <50 base pair deletion in the cytoplasmic region of the IgA allele (IgA tail-mutant; IgAtm/tm) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA-/-). IgA-/- exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4+ T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca2+ biosensor showed that IgA-/- had elevated Ca2+ signalling in Peyer's patch B cells. On the other hand, IgAtm/tm seemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder. CONCLUSION: IgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum.

Heterozygous mutation of the splicing factor Sf3b4 affects development of the axial skeleton and forebrain in mouse.

BACKGROUND: Splicing factor 3B subunit 4 (SF3B4) is a causative gene of an acrofacial dysostosis, Nager syndrome. Although in vitro analyses of SF3B4 have proposed multiple noncanonical functions unrelated to splicing, less information is available based on in vivo studies using model animals. RESULTS: We performed expression and functional analyses of Sf3b4 in mice. The mouse Sf3b4 transcripts were found from two-cell stage, and were ubiquitously present during embryogenesis with high expression levels in several tissues such as forming craniofacial bones and brain. In contrast, expression of a pseudogene-like sequence of mouse Sf3b4 (Sf3b4_ps) found by in silico survey was not detected up to embryonic day 10. We generated a Sf3b4 knockout mouse using CRISPR-Cas9 system. The homozygous mutant mouse of Sf3b4 was embryonic lethal. The heterozygous mutant of Sf3b4 mouse (Sf3b4+/- ) exhibited smaller body size compared to the wild-type from postnatal to adult period, as well as homeotic posteriorization of the vertebral morphology and flattened calvaria. The flattened calvaria appears to be attributable to mild microcephaly due to a lower cell proliferation rate in the forebrain. CONCLUSIONS: Our study suggests that Sf3b4 controls anterior-posterior patterning of the axial skeleton and guarantees cell proliferation for forebrain development in mice.

Visualization of Probiotic-Mediated Ca2+ Signaling in Intestinal Epithelial Cells In Vivo.

Probiotics, such as lactic acid bacteria (LAB) and Bacillus subtilis var. natto, have been shown to modulate immune responses. It is important to understand how probiotic bacteria impact intestinal epithelial cells (IECs), because IECs are the first line of defense at the mucosal surface barrier and their activities substantially affect the gut microenvironment and immunity. However, to date, their precise mechanism remains unknown due to a lack of analytical systems available for live animal models. Recently, we generated a conditional Ca2+ biosensor Yellow Cameleon (YC3.60) transgenic mouse line and established 5D (x, y, z, time, and Ca2+) intravital imaging systems of lymphoid tissues including those in Peyer's patches and bone marrow. In the present study, we further advance our intravital imaging system for intestinal tracts to visualize IEC responses against orally administrated food compounds in real time. Using this system, heat-killed B. subtilis natto, a probiotic TTCC012 strain, is shown to directly induce Ca2+ signaling in IECs in mice housed under specific pathogen-free conditions. In contrast, this activation is not observed in the Lactococcus lactis strain C60; however, when we generate germ-free YC3.60 mice and observe the LAB stimulation of IECs in the absence of gut microbiota, C60 is capable of inducing Ca2+ signaling. This is the first study to successfully visualize the direct effect of probiotics on IECs in live animals. These data strongly suggest that probiotic strains stimulate IECs under physiological conditions and that their activity is affected by the microenvironment of the small intestine, such as commensal bacteria.

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.

BACKGROUND: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. RESULTS: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. CONCLUSIONS: Our results provide a technical platform for high-throughput knock-in.

Intravital imaging of Ca(2+) signals in lymphocytes of Ca(2+) biosensor transgenic mice: indication of autoimmune diseases before the pathological onset.

Calcium ion (Ca(2+)) signaling is a typical phenomenon mediated through immune receptors, such as the B-cell antigen receptor (BCR), and it is important for their biological activities. To analyze the signaling of immune receptors together with their in vivo dynamics, we generated stable transgenic mice with the Föster/fluorescence resonance energy transfer (FRET)-based Ca(2+) indicator yellow cameleon 3.60 (YC3.60), based on the Cre/loxP system (YC3.60(flox)). We successfully obtained mice with specific YC3.60 expression in immune or nerve cells as well as mice with ubiquitous expression of this indicator. We established five-dimensional (5D) (x, y, z, time, and Ca(2+)) intravital imaging of lymphoid tissues, including the bone marrow. Furthermore, in autoimmune-prone models, the CD22(-/-) and C57BL/6- lymphoproliferation (lpr)/lpr mouse, Ca(2+) fluxes were augmented, although they did not induce autoimmune disease. Intravital imaging of Ca(2+) signals in lymphocytes may improve assessment of the risk of autoimmune diseases in model animals.

Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.

The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice.

Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools.

A global in vivo Drosophila RNAi screen identifies NOT3 as a conserved regulator of heart function.

Heart diseases are the most common causes of morbidity and death in humans. Using cardiac-specific RNAi-silencing in Drosophila, we knocked down 7061 evolutionarily conserved genes under conditions of stress. We present a first global roadmap of pathways potentially playing conserved roles in the cardiovascular system. One critical pathway identified was the CCR4-Not complex implicated in transcriptional and posttranscriptional regulatory mechanisms. Silencing of CCR4-Not components in adult Drosophila resulted in myofibrillar disarray and dilated cardiomyopathy. Heterozygous not3 knockout mice showed spontaneous impairment of cardiac contractility and increased susceptibility to heart failure. These heart defects were reversed via inhibition of HDACs, suggesting a mechanistic link to epigenetic chromatin remodeling. In humans, we show that a common NOT3 SNP correlates with altered cardiac QT intervals, a known cause of potentially lethal ventricular tachyarrhythmias. Thus, our functional genome-wide screen in Drosophila can identify candidates that directly translate into conserved mammalian genes involved in heart function.

Activating transcription factor 3 constitutes a negative feedback mechanism that attenuates saturated Fatty acid/toll-like receptor 4 signaling and macrophage activation in obese adipose tissue.

Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid-stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor-alpha production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor-alpha promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKA(y) mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation.

Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality.

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites. To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 (paternally expressed 10), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.

Autoamplification of NFATc1 expression determines its essential role in bone homeostasis.

NFATc1 and NFATc2 are functionally redundant in the immune system, but it was suggested that NFATc1 is required exclusively for differentiation of osteoclasts in the skeletal system. Here we provide genetic evidence that NFATc1 is essential for osteoclast differentiation in vivo by adoptive transfer of NFATc1(-/-) hematopoietic stem cells to osteoclast-deficient Fos(-/-) mice, and by Fos(-/-) blastocyst complementation, thus avoiding the embryonic lethality of NFATc1(-/-) mice. However, in vitro osteoclastogenesis in NFATc1-deficient cells was rescued by ectopic expression of NFATc2. The discrepancy between the in vivo essential role of NFATc1 and the in vitro effect of NFATc2 was attributed to selective autoregulation of the NFATc1 gene by NFAT through its promoter region. This suggested that an epigenetic mechanism contributes to the essential function of NFATc1 in cell lineage commitment. Thus, this study establishes that NFATc1 represents a potential therapeutic target for bone disease and reveals a mechanism that underlies the essential role of NFATc1 in bone homeostasis.