Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out (DKO) mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis.

MAST4 regulates stem cell maintenance with DLX3 for epithelial development and amelogenesis.

The asymmetric division of stem cells permits the maintenance of the cell population and differentiation for harmonious progress. Developing mouse incisors allows inspection of the role of the stem cell niche to provide specific insights into essential developmental phases. Microtubule-associated serine/threonine kinase family member 4 (Mast4) knockout (KO) mice showed abnormal incisor development with low hardness, as the size of the apical bud was decreased and preameloblasts were shifted to the apical side, resulting in amelogenesis imperfecta. In addition, Mast4 KO incisors showed abnormal enamel maturation, and stem cell maintenance was inhibited as amelogenesis was accelerated with Wnt signal downregulation. Distal-Less Homeobox 3 (DLX3), a critical factor in tooth amelogenesis, is considered to be responsible for the development of amelogenesis imperfecta in humans. MAST4 directly binds to DLX3 and induces phosphorylation at three residues within the nuclear localization site (NLS) that promotes the nuclear translocation of DLX3. MAST4-mediated phosphorylation of DLX3 ultimately controls the transcription of DLX3 target genes, which are carbonic anhydrase and ion transporter genes involved in the pH regulation process during ameloblast maturation. Taken together, our data reveal a novel role for MAST4 as a critical regulator of the entire amelogenesis process through its control of Wnt signaling and DLX3 transcriptional activity.

B Cells of Early-life Origin Defined by RAG2-based Lymphoid Cell Tracking under Native Hematopoietic Conditions.

During the perinatal period, the immune system sets the threshold to select either response or tolerance to environmental Ags, which leads to the potential to provide a lifetime of protection and health. B-1a B cells have been demonstrated to develop during this perinatal time window, showing a unique and restricted BCR repertoire, and these cells play a major role in natural Ab secretion and immune regulation. In the current study, we developed a highly efficient temporally controllable RAG2-based lymphoid lineage cell labeling and tracking system and applied this system to understand the biological properties and contribution of B-1a cells generated at distinct developmental periods to the adult B-1a compartments. This approach revealed that B-1a cells with a history of RAG2 expression during the embryonic and neonatal periods dominate the adult B-1a compartment, including those in the bone marrow (BM), peritoneal cavity, and spleen. Moreover, the BCR repertoire of B-1a cells with a history of RAG2 expression during the embryonic period was restricted, becoming gradually more diverse during the neonatal period, and then heterogeneous at the adult stage. Furthermore, more than half of plasmablasts/plasma cells in the adult BM had embryonic and neonatal RAG2 expression histories. Moreover, BCR analysis revealed a high relatedness between BM plasmablasts/plasma cells and B-1a cells derived from embryonic and neonatal periods, suggesting that these cell types have a common origin. Taken together, these findings define, under native hematopoietic conditions, the importance in adulthood of B-1a cells generated during the perinatal period.

A rare olive compound oleacein functions as a TrkB agonist and mitigates neuroinflammation both in vitro and in vivo.

BACKGROUND: Neuroinflammation is widely acknowledged as a characteristic feature of almost all neurological disorders and specifically in depression- and anxiety-like disorders. In recent years, there has been significant attention on natural compounds with potent anti-inflammatory effects due to their potential in mitigating neuroinflammation and neuroplasticity. METHODS: In the present study, we aimed to evaluate the neuroprotective effects of oleacein (OC), a rare secoiridoid derivative found in extra virgin olive oil. Our goal was to explore the BDNF/TrkB neurotrophic activity of OC and subsequently assess its potential for modulating neuroinflammatory response using human neuroblastoma cells (SH-SY5Y cells) and an in vivo model of depression induced by lipopolysaccharide (LPS)-mediated inflammation. RESULTS: In SH-SY5Y cells, OC exhibited a significant dose-dependent increase in BDNF expression. This enhancement was absent when cells were co-treated with inhibitors of BDNF's receptor TrkB, as well as downstream molecules PI3K and MEK. Whole-transcriptomics analysis revealed that OC upregulated cell cycle-related genes under normal conditions, while downregulating inflammation-associated genes in LPS-induced conditions. Furthermore, surface plasmon resonance (SPR) assays demonstrated that OC exhibited a stronger and more stable binding affinity to TrkB compared to the positive control, 7,8-dihydroxyflavone. Importantly, bioluminescence imaging revealed that a single oral dose of OC significantly increased BDNF expression in the brains of Bdnf-IRES-AkaLuc mice. Furthermore, oral administration of OC at a dosage of 10 mg/kg body weight for 10 days significantly reduced immobility time in the tail suspension test compared to the LPS-treated group. RT-qPCR analysis revealed that OC significantly decreased the expression of pro-inflammatory cytokines Tnfα, Il6, and Il1ß, while simultaneously enhancing Bdnf expression, as well as both pro and mature BDNF protein levels in mice hippocampus. These changes were comparable to those induced by the positive control antidepressant drug fluoxetine. Additionally, microarray analysis of mouse brains confirmed that OC could counteract LPS-induced inflammatory biological events. CONCLUSION: Altogether, our study represents the first report on the potential antineuroinflammatory and antidepressant properties of OC via modulation of BDNF/TrkB neurotrophic activity. This finding underscores the potential of OC as a natural therapeutic agent for depression- and anxiety-related disorders.

Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions.

Functional regions that regulate biological phenomena are interspersed throughout eukaryotic genomes. The most definitive approach for identifying such regions is to confirm the phenotype of cells or organisms in which specific regions have been mutated or removed from the genome. This approach is invaluable for the functional analysis of genes with a defined functional element, the protein-coding sequence. By contrast, no functional analysis platforms have been established for the study of cis-elements or microRNA cluster regions consisting of multiple microRNAs with functional overlap. Whole-genome mutagenesis approaches, such as via N-ethyl-N-nitrosourea and gene trapping, have greatly contributed to elucidating the function of coding genes. These methods almost never induce deletions of genomic regions or multiple mutations within a narrow region. In other words, cis-elements and microRNA clusters cannot be effectively targeted in such a manner. Herein, we established a novel region-specific random mutagenesis method named CRISPR- and transposase-based regional mutagenesis (CTRL-mutagenesis). We demonstrate that CTRL-mutagenesis randomly induces diverse mutations within target regions in murine embryonic stem cells. Comparative analysis of mutants harbouring subtly different mutations within the same region would facilitate the further study of cis-element and microRNA clusters.

Impaired Glycosylation of Gastric Mucins Drives Gastric Tumorigenesis and Serves as a Novel Therapeutic Target.

BACKGROUND & AIMS: Gastric cancer is often accompanied by a loss of mucin 6 (MUC6), but its pathogenic role in gastric carcinogenesis remains unclear. METHODS: Muc6 knockout (Muc6-/-) mice and Muc6-dsRED mice were newly generated. Tff1Cre, Golph3-/-, R26-Golgi-mCherry, Hes1flox/flox, Cosmcflox/flox, and A4gnt-/- mice were also used. Histology, DNA and RNA, proteins, and sugar chains were analyzed by whole-exon DNA sequence, RNA sequence, immunohistochemistry, lectin-binding assays, and liquid chromatography-mass spectrometry analysis. Gastric organoids and cell lines were used for in vitro assays and xenograft experiments. RESULTS: Deletion of Muc6 in mice spontaneously causes pan-gastritis and invasive gastric cancers. Muc6-deficient tumor growth was dependent on mitogen-activated protein kinase activation, mediated by Golgi stress-induced up-regulation of Golgi phosphoprotein 3. Glycomic profiling revealed aberrant expression of mannose-rich N-linked glycans in gastric tumors, detected with banana lectin in association with lack of MUC6 expression. We identified a precursor of clusterin as a binding partner of mannose glycans. Mitogen-activated protein kinase activation, Golgi stress responses, and aberrant mannose expression are found in separate Cosmc- and A4gnt-deficient mouse models that lack normal O-glycosylation. Banana lectin-drug conjugates proved an effective treatment for mannose-rich murine and human gastric cancer. CONCLUSIONS: We propose that Golgi stress responses and aberrant glycans are important drivers of and promising new therapeutic targets for gastric cancer.

Landscape of driver mutations and their clinical effects on Down syndrome-related myeloid neoplasms.

ABSTRACT: Transient abnormal myelopoiesis (TAM) is a common complication in newborns with Down syndrome (DS). It commonly progresses to myeloid leukemia (ML-DS) after spontaneous regression. In contrast to the favorable prognosis of primary ML-DS, patients with refractory/relapsed ML-DS have poor outcomes. However, the molecular basis for refractoriness and relapse and the full spectrum of driver mutations in ML-DS remain largely unknown. We conducted a genomic profiling study of 143 TAM, 204 ML-DS, and 34 non-DS acute megakaryoblastic leukemia cases, including 39 ML-DS cases analyzed by exome sequencing. Sixteen novel mutational targets were identified in ML-DS samples. Of these, inactivations of IRX1 (16.2%) and ZBTB7A (13.2%) were commonly implicated in the upregulation of the MYC pathway and were potential targets for ML-DS treatment with bromodomain-containing protein 4 inhibitors. Partial tandem duplications of RUNX1 on chromosome 21 were also found, specifically in ML-DS samples (13.7%), presenting its essential role in DS leukemia progression. Finally, in 177 patients with ML-DS treated following the same ML-DS protocol (the Japanese Pediatric Leukemia and Lymphoma Study Group acute myeloid leukemia -D05/D11), CDKN2A, TP53, ZBTB7A, and JAK2 alterations were associated with a poor prognosis. Patients with CDKN2A deletions (n = 7) or TP53 mutations (n = 4) had substantially lower 3-year event-free survival (28.6% vs 90.5%; P < .001; 25.0% vs 89.5%; P < .001) than those without these mutations. These findings considerably change the mutational landscape of ML-DS, provide new insights into the mechanisms of progression from TAM to ML-DS, and help identify new therapeutic targets and strategies for ML-DS.

Engineered FSHD mutations results in D4Z4 heterochromatin disruption and feedforward DUX4 network activation.

Facioscapulohumeral dystrophy (FSHD) is linked to contraction of D4Z4 repeats on chromosome 4q with SMCHD1 mutations acting as a disease modifier. D4Z4 heterochromatin disruption and abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, are the hallmarks of FSHD. However, defining the precise effect of D4Z4 contraction has been difficult because D4Z4 repeats are primate-specific and DUX4 expression is very rare in highly heterogeneous patient myocytes. We generated isogenic mutant cell lines harboring D4Z4 and/or SMCHD1 mutations in a healthy human skeletal myoblast line. We found that the mutations affect D4Z4 heterochromatin differently, and that SMCHD1 mutation or disruption of DNA methylation stabilizes otherwise variegated DUX4 target activation in D4Z4 contraction mutant cells, demonstrating the critical role of modifiers. Our study revealed amplification of the DUX4 signal through downstream targets, H3.X/Y and LEUTX. Our results provide important insights into how rare DUX4 expression leads to FSHD pathogenesis.

MAX controls meiotic entry in sexually undifferentiated germ cells.

Meiosis is a specialized type of cell division that occurs physiologically only in germ cells. We previously demonstrated that MYC-associated factor X (MAX) blocks the ectopic onset of meiosis in embryonic and germline stem cells in culture systems. Here, we investigated the Max gene's role in mouse primordial germ cells. Although Max is generally ubiquitously expressed, we revealed that sexually undifferentiated male and female germ cells had abundant MAX protein because of their higher Max gene expression than somatic cells. Moreover, our data revealed that this high MAX protein level in female germ cells declined significantly around physiological meiotic onset. Max disruption in sexually undifferentiated germ cells led to ectopic and precocious expression of meiosis-related genes, including Meiosin, the gatekeeper of meiotic onset, in both male and female germ cells. However, Max-null male and female germ cells did not complete the entire meiotic process, but stalled during its early stages and were eventually eliminated by apoptosis. Additionally, our meta-analyses identified a regulatory region that supports the high Max expression in sexually undifferentiated male and female germ cells. These results indicate the strong connection between the Max gene and physiological onset of meiosis in vivo through dynamic alteration of its expression.

SERTAD1 initiates NLRP3-mediated inflammasome activation through restricting NLRP3 polyubiquitination.

We here demonstrate that SERTAD1 is an adaptor protein responsible for the regulation of lysine 63 (K63)-linked NLRP3 polyubiquitination by the Cullin1 E3 ubiquitin ligase upon inflammasome activation. SERTAD1 specifically binds to NLRP3 but not to other inflammasome sensors. This endogenous interaction increases after inflammasome activation, interfering with the interaction between NLRP3 and Cullin1. Interleukin (IL)-1ß and IL-18 secretion, as well as the cleavage of gasdermin D, are decreased in SERTAD1 knockout bone-marrow-derived macrophages, together with reduced formation of the NLRP3 inflammasome complex. Additionally, SERTAD1-deficient mice show attenuated severity of monosodium-uric-acid-induced peritonitis and experimental autoimmune encephalomyelitis. Analysis of public datasets indicates that expression of SERTAD1 mRNA is significantly increased in the patients of autoimmune diseases. Thus, our findings uncover a function of SERTAD1 that specifically reduces Cullin1-mediated NLRP3 polyubiquitination via direct binding to NLRP3, eventually acting as a crucial factor to regulate the initiation of NLRP3-mediated inflammasome activation.

Deletion of Exoc7, but not Exoc3, in male germ cells causes severe spermatogenesis failure with spermatocyte aggregation in mice.

Vesicular trafficking is essential for the transport of intracellularly produced functional molecules to the plasma membrane and extracellular space. The exocyst complex, composed of eight different proteins, is an important functional machinery for "tethering" in vesicular trafficking. Functional studies have been conducted in laboratory mice to identify the mechanisms by which the deletion of each exocyst factor affect various biological phenomena. Interestingly, each exocyst factor-deficient mutant exhibits a different phenotype. This discrepancy may be due to the function of the exocyst factor beyond its role as a component of the exocyst complex. Male germline-specific conditional knockout (cKO) mice of the Exoc1 gene, which encodes one of the exocyst factors EXOC1 (SEC3), exhibit severe spermatogenesis defects; however, whether this abnormality also occurs in mutants lacking other exocyst factors remains unknown. In this study, we found that exocyst factor EXOC3 (SEC6) was not required for spermatogenesis, but depletion of EXOC7 (EXO70) led to severe spermatogenesis defects. In addition to being a component of the exocyst complex, EXOC1 has other functions. Notably, male germ cell-specific Exoc7 cKO and Exoc1 cKO mice exhibited phenotypic similarities, suggesting the importance of the exocyst complex for spermatogenesis. The results of this study will contribute to further understanding of spermatogenesis from the aspect of vesicular trafficking.

Role of inter-alpha-trypsin inhibitor heavy chain 4 and its citrullinated form in experimental arthritis murine models.

Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is a major protein in serum and reported to be upregulated at the onset of rheumatoid arthritis (RA). Its citrullinated form, cit-ITIH4, is specifically found in the serum and synovial fluid of patients with RA. However, the detailed function of ITIH4 in arthritis remains unknown. The aim of this study was to clarify the role of ITIH4 and cit-ITIH4 using experimental arthritis models. ITIH4 and cit-ITIH4 expression was examined in steady-state mice and two different arthritis models, and their pathological effects were examined in Itih4-deficient mice. In naïve C57BL/6 (WT) mice, ITIH4 was expressed as mRNA in the liver and the lung and was expressed as protein in serum and hepatocytes. In K/BxN serum transferred arthritis (K/BxN-STA) and collagen-induced arthritis (CIA), ITIH4 and cit-ITIH4 in sera were increased before the onset of arthritis, and cit-ITIH4 was further increased at the peak of arthritis. In Itih4-deficient mice, citrullinated proteins in serum and joints, especially 120 kDa protein, were clearly diminished; however, there was no significant difference in arthritis severity between WT and itih-/- mice either in the K/BxN-STA or CIA model. CIA mice also exhibited pulmonary lesions and itih4-/- mice tended to show enhanced inflammatory cell aggregation compared to WT mice. Neutrophils in the lungs of itih4-/- mice were significantly increased compared to WT mice. In summary, ITIH4 itself did not alter the severity of arthritis but may inhibit autoimmune inflammation via suppression of neutrophil recruitment.

Establishment and visual analysis of CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice.

In CBA/J and C3H/HeJ mice, retinitis pigmentosa is inherited as an autosomal-recessive trait due to a mutation in Pde6b, which encodes cGMP phosphodiesterase subunit b. In these strains, the Y347X mutation in Pde6b leads to the upregulation of cGMP levels, increased Ca2+ influx induces rod death, and the outer segment and rod cells entirely disappeared by 35 days after birth. In the present study, we utilized the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9-mediated gene editing to repair the Y347X mutation in CBA/J and C3H/HeJ mice. Evaluation of the established CBA/J-Pde6bY347Y/Y347X and C3H/HeJ-Pde6bY347Y/Y347X mice, which were confirmed to have normal retinal layers by live fundoscopic imaging and histopathological analysis, revealed improved visual acuity based on the visual cliff and light/dark latency tests. Furthermore, our analyses revealed that the visible platform test was a more effective tool for testing visual behavior in these mice. The results suggest that the established strains can serve as control groups for CBA/J and C3H/HeJ in ophthalmology studies involving retinitis pigmentosa.

Tm4sf19 deficiency inhibits osteoclast multinucleation and prevents bone loss.

BACKGROUND: Multinucleation is a hallmark of osteoclast formation and has a unique ability to resorb bone matrix. During osteoclast differentiation, the cytoskeleton reorganization results in the generation of actin belts and eventual bone resorption. Tetraspanins are involved in adhesion, migration and fusion in various cells. However, its function in osteoclast is still unclear. In this study, we identified Tm4sf19, a member of the tetraspanin family, as a regulator of osteoclast function. MATERIALS AND METHODS: We investigate the effect of Tm4sf19 deficiency on osteoclast differentiation using bone marrow-derived macrophages obtained from wild type (WT), Tm4sf19 knockout (KO) and Tm4sf19 LELΔ mice lacking the large extracellular loop (LEL). We analyzed bone mass of young and aged WT, KO and LELΔ mice by µCT analysis. The effects of Tm4sf19 LEL-Fc fusion protein were accessed in osteoclast differentiation and osteoporosis animal model. RESULTS: We found that deficiency of Tm4sf19 inhibited osteoclast function and LEL of Tm4sf19 was responsible for its function in osteoclasts in vitro. KO and LELΔ mice exhibited higher trabecular bone mass compared to WT mice. We found that Tm4sf19 interacts with integrin αvß3 through LEL, and that this binding is important for cytoskeletal rearrangements in osteoclast by regulating signaling downstream of integrin αvß3. Treatment with LEL-Fc fusion protein inhibited osteoclast function in vitro and administration of LEL-Fc prevented bone loss in an osteoporosis mouse model in vivo. CONCLUSION: We suggest that Tm4sf19 regulates osteoclast function and that LEL-Fc may be a promising drug to target bone destructive diseases caused by osteoclast hyper-differentiation.

Null mutation of exocyst complex component 3-like does not affect vascular development in mice.

Exocyst is an octameric protein complex implicated in exocytosis. The exocyst complex is highly conserved among mammalian species, but the physiological function of each subunit in exocyst remains unclear. Previously, we identified exocyst complex component 3-like (Exoc3l) as a gene abundantly expressed in embryonic endothelial cells and implicated in the process of angiogenesis in human umbilical cord endothelial cells. Here, to reveal the physiological roles of Exoc3l during development, we generated Exoc3l knockout (KO) mice by genome editing with CRISPR/Cas9. Exoc3l KO mice were viable and showed no significant phenotype in embryonic angiogenesis or postnatal retinal angiogenesis. Exoc3l KO mice also showed no significant alteration in cholesterol homeostasis or insulin secretion, although several reports suggest an association of Exoc3l with these processes. Despite the implied roles, Exoc3l KO mice exhibited no apparent phenotype in vascular development, cholesterol homeostasis, or insulin secretion.

Intestinal Atp8b1 dysfunction causes hepatic choline deficiency and steatohepatitis.

Choline is an essential nutrient, and its deficiency causes steatohepatitis. Dietary phosphatidylcholine (PC) is digested into lysoPC (LPC), glycerophosphocholine, and choline in the intestinal lumen and is the primary source of systemic choline. However, the major PC metabolites absorbed in the intestinal tract remain unidentified. ATP8B1 is a P4-ATPase phospholipid flippase expressed in the apical membrane of the epithelium. Here, we use intestinal epithelial cell (IEC)-specific Atp8b1-knockout (Atp8b1IEC-KO) mice. These mice progress to steatohepatitis by 4 weeks. Metabolomic analysis and cell-based assays show that loss of Atp8b1 in IEC causes LPC malabsorption and thereby hepatic choline deficiency. Feeding choline-supplemented diets to lactating mice achieves complete recovery from steatohepatitis in Atp8b1IEC-KO mice. Analysis of samples from pediatric patients with ATP8B1 deficiency suggests its translational potential. This study indicates that Atp8b1 regulates hepatic choline levels through intestinal LPC absorption, encouraging the evaluation of choline supplementation therapy for steatohepatitis caused by ATP8B1 dysfunction.

Activity of estrogen receptor ß expressing neurons in the medial amygdala regulates preference toward receptive females in male mice.

The processing of information regarding the sex and reproductive state of conspecific individuals is critical for successful reproduction and survival in males. Generally, male mice exhibit a preference toward the odor of sexually receptive (RF) over nonreceptive females (XF) or gonadally intact males (IM). Previous studies suggested the involvement of estrogen receptor beta (ERß) expressed in the medial amygdala (MeA) in male preference toward RF. To further delineate the role played by ERß in the MeA in the neuronal network regulating male preference, we developed a new ERß-iCre mouse line using the CRISPR-Cas9 system. Fiber photometry Ca2+ imaging revealed that ERß-expressing neurons in the postero-dorsal part of the MeA (MeApd-ERß+ neurons) were more active during social investigation toward RF compared to copresented XF or IM mice in a preference test. Chemogenetic inhibition of MeApd-ERß+ neuronal activity abolished a preference to RF in "RF vs. XF," but not "RF vs. IM," tests. Analysis with cre-dependent retrograde tracing viral vectors identified the principal part of the bed nucleus of stria terminalis (BNSTp) as a primary projection site of MeApd-ERß+ neurons. Fiber photometry recording in the BNSTp during a preference test revealed that chemogenetic inhibition of MeApd-ERß+ neurons abolished differential neuronal activity of BNSTp cells as well as a preference to RF against XF but not against IM mice. Collectively, these findings demonstrate for the first time that MeApd-ERß+ neuronal activity is required for expression of receptivity-based preference (i.e., RF vs. XF) but not sex-based preference (i.e., RF vs. IM) in male mice.

Development of two mouse strains conditionally expressing bright luciferases with distinct emission spectra as new tools for in vivo imaging.

In vivo bioluminescence imaging (BLI) has been an invaluable noninvasive method to visualize molecular and cellular behaviors in laboratory animals. Bioluminescent reporter mice harboring luciferases for general use have been limited to a classical luciferase, Luc2, from Photinus pyralis, and have been extremely powerful for various in vivo studies. However, applicability of reporter mice for in vivo BLI could be further accelerated by increasing light intensity through the use of other luciferases and/or by improving the biodistribution of their substrates in the animal body. Here we created two Cre-dependent reporter mice incorporating luciferases oFluc derived from Pyrocoeli matsumurai and Akaluc, both of which had been reported previously to be brighter than Luc2 when using appropriate substrates; we then tested their bioluminescence in neural tissues and other organs in living mice. When expressed throughout the body, both luciferases emitted an intense yellow (oFluc) or far-red (Akaluc) light easily visible to the naked eye. oFluc and Akaluc were similarly bright in the pancreas for in vivo BLI; however, Akaluc was superior to oFluc for brain imaging, because its substrate, AkaLumine-HCl, was distributed to the brain more efficiently than the oFluc substrate, D-luciferin. We also demonstrated that the lights produced by oFluc and Akaluc were sufficiently spectrally distinct from each other for dual-color imaging in a single living mouse. Taken together, these novel bioluminescent reporter mice are an ideal source of cells with bright bioluminescence and may facilitate in vivo BLI of various tissues/organs for preclinical and biomedical research in combination with a wide variety of Cre-driver mice.

A universal method for generating knockout mice in multiple genetic backgrounds using zygote electroporation.

Genetically engineered mouse models are essential tools for understanding mammalian gene functions and disease pathogenesis. Genome editing allows the generation of these models in multiple inbred strains of mice without backcrossing. Zygote electroporation dramatically removed the barrier for introducing the CRISPR-Cas9 complex in terms of cost and labour. Here, we demonstrate that the generalised zygote electroporation method is also effective for generating knockout mice in multiple inbred strains. By combining in vitro fertilisation and electroporation, we obtained founders for knockout alleles in eight common inbred strains. Long-read sequencing analysis detected not only intended mutant alleles but also differences in read frequency of intended and unintended alleles among strains. Successful germline transmission of knockout alleles demonstrated that our approach can establish mutant mice targeting the same locus in multiple inbred strains for phenotyping analysis, contributing to reverse genetics and human disease research.

Loss of CtBP2 may be a mechanistic link between metabolic derangements and progressive impairment of pancreatic ß cell function.

The adaptive increase in insulin secretion in early stages of obesity serves as a safeguard mechanism to maintain glucose homeostasis that cannot be sustained, and the eventual decompensation of ß cells is a key event in the pathogenesis of diabetes. Here we describe a crucial system orchestrated by a transcriptional cofactor CtBP2. In cultured ß cells, insulin gene expression is coactivated by CtBP2. Global genomic mapping of CtBP2 binding sites identifies a key interaction between CtBP2 and NEUROD1 through which CtBP2 decompacts chromatin in the insulin gene promoter. CtBP2 expression is diminished in pancreatic islets in multiple mouse models of obesity, as well as human obesity. Pancreatic ß cell-specific CtBP2-deficient mice manifest glucose intolerance with impaired insulin secretion. Our transcriptome analysis highlights an essential role of CtBP2 in the maintenance of ß cell integrity. This system provides clues to the molecular basis in obesity and may be targetable to develop therapeutic approaches.