Myeloid cell-specific ablation of Runx2 gene exacerbates post-infarct cardiac remodeling.

Runt-related transcription factor 2 (Runx2), a regulator of osteoblast differentiation, is pathologically involved in vascular calcification; however, the significance of Runx2 in cardiac homeostasis remains unclear. Here, we investigated the roles of Runx2 in cardiac remodeling after myocardial infarction (MI). The expression of Runx2 mRNA and protein was upregulated in murine hearts after MI. Runx2 was expressed in heart-infiltrating myeloid cells, especially in macrophages, at the border zone of post-infarct myocardium. To analyze the biological functions of Runx2 in cardiac remodeling, myeloid cell-specific Runx2 deficient (CKO) mice were exposed to MI. After MI, ventricular weight/tibia length ratio was increased in CKO mice, concomitant with severe cardiac dysfunction. Cardiac fibrosis was exacerbated in CKO mice, consistent with the upregulation of collagen 1a1 expression. Mechanistically, immunohistochemical analysis using anti-CD31 antibody showed that capillary density was decreased in CKO mice. Additionally, conditioned culture media of myeloid cells from Runx2 deficient mice exposed to MI induced the tube formation of vascular endothelial cells to a lesser extent than those from control mice. RNA-sequence showed that the expression of pro-angiogenic or anti-angiogenic factors was altered in macrophages from Runx2-deficient mice. Collectively, Runx2+ myeloid cells infiltrate into post-infarct myocardium and prevent adverse cardiac remodeling, at least partially, by regulating endothelial cell function.

Deficiency of lung-specific claudin-18 leads to aggravated infection with Cryptococcus deneoformans through dysregulation of the microenvironment in lungs.

Cryptococcus deneoformans is an opportunistic fungal pathogen that infects the lungs via airborne transmission and frequently causes fatal meningoencephalitis. Claudins (Cldns), a family of proteins with 27 members found in mammals, form the tight junctions within epithelial cell sheets. Cldn-4 and 18 are highly expressed in airway tissues, yet the roles of these claudins in respiratory infections have not been clarified. In the present study, we analyzed the roles of Cldn-4 and lung-specific Cldn-18 (luCldn-18) in host defense against C. deneoformans infection. luCldn-18-deficient mice exhibited increased susceptibility to pulmonary infection, while Cldn-4-deficient mice had normal fungal clearance. In luCldn-18-deficient mice, production of cytokines including IFN-γ was significantly decreased compared to wild-type mice, although infiltration of inflammatory cells including CD4+ T cells into the alveolar space was significantly increased. In addition, luCldn-18 deficiency led to high K+ ion concentrations in bronchoalveolar lavage fluids and also to alveolus acidification. The fungal replication was significantly enhanced both in acidic culture conditions and in the alveolar spaces of luCldn-18-deficient mice, compared with physiological pH conditions and those of wild-type mice, respectively. These results suggest that luCldn-18 may affect the clinical course of cryptococcal infection indirectly through dysregulation of the alveolar space microenvironment.

Targeted gene disruption in a marsupial, Monodelphis domestica, by CRISPR/Cas9 genome editing.

Marsupials represent one of three extant mammalian subclasses with very unique characteristics not shared by other mammals. Most notably, much of the development of neonates immaturely born after a relatively short gestation takes place in the external environment. Among marsupials, the gray short-tailed opossum (Monodelphis domestica; hereafter "the opossum") is one of very few established laboratory models. Due to many biologically unique characteristics and experimentally advantageous features, the opossum is used as a prototype species for basic research on marsupial biology.1,2 However, in vivo studies of gene function in the opossum, and thus marsupials in general, lag far behind those of eutherian mammals due to the lack of reliable means to manipulate their genomes. In this study, we describe the successful generation of genome edited opossums by a combination of refined methodologies in reproductive biology and embryo manipulation. We took advantage of the opossum's resemblance to popular rodent models, such as the mouse and rat, in body size and breeding characteristics. First, we established a tractable pipeline of reproductive technologies, from induction of ovulation, timed copulation, and zygote collection to embryo transfer to pseudopregnant females, that warrant an essential platform to manipulate opossum zygotes. Further, we successfully demonstrated the generation of gene knockout opossums at the Tyr locus by microinjection of pronuclear stage zygotes using CRISPR/Cas9 genome editing, along with germline transmission of the edited alleles to the F1 generation. This study provides a critical foundation for venues to expand mammalian reverse genetics into the metatherian subclass.

CAMSAP1 breaks the homeostatic microtubule network to instruct neuronal polarity.

The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.

Prc1-rich kinetochores are required for error-free acentrosomal spindle bipolarization during meiosis I in mouse oocytes.

Acentrosomal meiosis in oocytes represents a gametogenic challenge, requiring spindle bipolarization without predefined bipolar cues. While much is known about the structures that promote acentrosomal microtubule nucleation, less is known about the structures that mediate spindle bipolarization in mammalian oocytes. Here, we show that in mouse oocytes, kinetochores are required for spindle bipolarization in meiosis I. This process is promoted by oocyte-specific, microtubule-independent enrichment of the antiparallel microtubule crosslinker Prc1 at kinetochores via the Ndc80 complex. In contrast, in meiosis II, cytoplasm that contains upregulated factors including Prc1 supports kinetochore-independent pathways for spindle bipolarization. The kinetochore-dependent mode of spindle bipolarization is required for meiosis I to prevent chromosome segregation errors. Human oocytes, where spindle bipolarization is reportedly error prone, exhibit no detectable kinetochore enrichment of Prc1. This study reveals an oocyte-specific function of kinetochores in acentrosomal spindle bipolarization in mice, and provides insights into the error-prone nature of human oocytes.

LUBAC accelerates B-cell lymphomagenesis by conferring resistance to genotoxic stress on B cells.

The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death and is a suitable therapeutic target for B-cell lymphomas.

Fhod1, an actin-organizing formin family protein, is dispensable for cardiac development and function in mice.

The formin family proteins have the ability to regulate actin filament assembly, thereby functioning in diverse cytoskeletal processes. Fhod3, a cardiac member of the family, plays a crucial role in development and functional maintenance of the heart. Although Fhod1, a protein closely-related to Fhod3, has been reported to be expressed in cardiomyocytes, the role of Fhod1 in the heart has still remained elusive. To know the physiological role of Fhod1 in the heart, we disrupted the Fhod1 gene in mice by replacement of exon 1 with a lacZ reporter gene. Histological lacZ staining unexpectedly revealed no detectable expression of Fhod1 in the heart, in contrast to intensive staining in the lung, a Fhod1-containing organ. Consistent with this, expression level of the Fhod1 protein in the heart was below the lower limit of detection of the present immunoblot analysis with three independent anti-Fhod1 antibodies. Homozygous Fhod1-null mice did not show any defects in gross and histological appearance of the heart or upregulate fetal cardiac genes that are induced under stress conditions. Furthermore, Fhod1 ablation did not elicit compensatory increase in expression of other formins. Thus, Fhod1 appears to be dispensable for normal development and function of the mouse heart, even if a marginal amount of Fhod1 is expressed in the heart.

The PTN-PTPRZ signal activates the AFAP1L2-dependent PI3K-AKT pathway for oligodendrocyte differentiation: Targeted inactivation of PTPRZ activity in mice.

Protein tyrosine phosphatase receptor type Z (PTPRZ) maintains oligodendrocyte precursor cells (OPCs) in an undifferentiated state. The inhibition of PTPase by its ligand pleiotrophin (PTN) promotes OPC differentiation; however, the substrate molecules of PTPRZ involved in the differentiation have not yet been elucidated in detail. We herein demonstrated that the tyrosine phosphorylation of AFAP1L2, paxillin, ERBB4, GIT1, p190RhoGAP, and NYAP2 was enhanced in OPC-like OL1 cells by a treatment with PTN. AFAP1L2, an adaptor protein involved in the PI3K-AKT pathway, exhibited the strongest response to PTN. PTPRZ dephosphorylated AFAP1L2 at tyrosine residues in vitro and in HEK293T cells. In OL1 cells, the knockdown of AFAP1L2 or application of a PI3K inhibitor suppressed cell differentiation as well as the PTN-induced phosphorylation of AKT and mTOR. We generated a knock-in mouse harboring a catalytically inactive Cys to Ser (CS) mutation in the PTPase domain. The phosphorylation levels of AFAP1L2, AKT, and mTOR were higher, and the expression of oligodendrocyte markers, including myelin basic protein (MBP) and myelin regulatory factor (MYRF), was stronger in CS knock-in brains than in wild-type brains on postnatal day 10; however, these differences mostly disappeared in the adult stage. Adult CS knock-in mice exhibited earlier remyelination after cuprizone-induced demyelination through the accelerated differentiation of OPCs. These phenotypes in CS knock-in mice were similar to those in Ptprz-deficient mice. Therefore, we conclude that the PTN-PTPRZ signal stimulates OPC differentiation partly by enhancing the tyrosine phosphorylation of AFAP1L2 in order to activate the PI3K-AKT pathway.

Deletion of Long Isoform of Eukaryotic Elongation Factor 1Bδ Leads to Audiogenic Seizures and Aversive Stimulus-Induced Long-Lasting Activity Suppression in Mice.

Alternative splicing enables a gene to give rise to diverse protein products. The Eef1d gene produces two isoforms: a short isoform that encodes translation elongation factor 1Bδ (eEF1Bδ1), and a long isoform that encodes the heat shock-responsive transcription factor eEF1BδL. Previously, we found that eEF1BδL was a splice variant that was specific to the brain and testis, and the protein encoded is thought to have a function in the central nervous system. In this study, we generated knockout (KO) mice of C57BL/6J background that selectively lacked a specific exon in Eef1d for the long isoform. These KO mice lacked eEF1BδL, but not eEF1Bδ1, in the brain. Although the KO mice showed normal anxiety-related and learning behavior in behavioral tests, some showed severe seizures in response to loud sounds (90 dBA), an audiogenic seizures (AGS) response. Furthermore, after the KO mice had been subjected to the fear conditioning test, they showed remarkably decreased locomotor activity in their home cage and in the open-field and elevated plus-maze tests. After the fear conditioning test, a significant decrease in brain weight, atrophy of the hippocampus and midbrain, and reduced cortical layer thickness were observed in the KO mice. We also found a compensatory increase in the eEF1Bδ1 level and elevated protein synthesis with the induction of endoplasmic reticulum stress markers in these mice. Our results suggest that eEF1BδL has an important role in normal brain function especially when exposed to external stimuli.