Noncanonical imprinting sustains embryonic development and restrains placental overgrowth.

Genomic imprinting regulates parental origin-dependent monoallelic gene expression. It is mediated by either germline differential methylation of DNA (canonical imprinting) or oocyte-derived H3K27me3 (noncanonical imprinting) in mice. Depletion of Eed, an essential component of Polycomb repressive complex 2, results in genome-wide loss of H3K27me3 in oocytes, which causes loss of noncanonical imprinting (LOI) in embryos. Although Eed maternal KO (matKO) embryos show partial lethality after implantation, it is unknown whether LOI itself contributes to the developmental phenotypes of these embryos, which makes it unclear whether noncanonical imprinting is developmentally relevant. Here, by combinatorial matKO of Xist, a noncanonical imprinted gene whose LOI causes aberrant transient maternal X-chromosome inactivation (XCI) at preimplantation, we show that prevention of the transient maternal XCI greatly restores the development of Eed matKO embryos. Moreover, we found that the placentae of Eed matKO embryos are remarkably enlarged in a manner independent of Xist LOI. Heterozygous deletion screening of individual autosomal noncanonical imprinted genes suggests that LOI of the Sfmbt2 miRNA cluster chromosome 2 miRNA cluster (C2MC), solute carrier family 38 member 4 (Slc38a4), and Gm32885 contributes to the placental enlargement. Taken together, our study provides evidence that Xist imprinting sustains embryonic development and that autosomal noncanonical imprinting restrains placental overgrowth.

Highly rigid H3.1/H3.2-H3K9me3 domains set a barrier for cell fate reprogramming in trophoblast stem cells.

The placenta is a highly evolved, specialized organ in mammals. It differs from other organs in that it functions only for fetal maintenance during gestation. Therefore, there must be intrinsic mechanisms that guarantee its unique functions. To address this question, we comprehensively analyzed epigenomic features of mouse trophoblast stem cells (TSCs). Our genome-wide, high-throughput analyses revealed that the TSC genome contains large-scale (>1-Mb) rigid heterochromatin architectures with a high degree of histone H3.1/3.2-H3K9me3 accumulation, which we termed TSC-defined highly heterochromatinized domains (THDs). Importantly, depletion of THDs by knockdown of CAF1, an H3.1/3.2 chaperone, resulted in down-regulation of TSC markers, such as Cdx2 and Elf5, and up-regulation of the pluripotent marker Oct3/4, indicating that THDs maintain the trophoblastic nature of TSCs. Furthermore, our nuclear transfer technique revealed that THDs are highly resistant to genomic reprogramming. However, when H3K9me3 was removed, the TSC genome was fully reprogrammed, giving rise to the first TSC cloned offspring. Interestingly, THD-like domains are also present in mouse and human placental cells in vivo, but not in other cell types. Thus, THDs are genomic architectures uniquely developed in placental lineage cells, which serve to protect them from fate reprogramming to stably maintain placental function.

Nesprin1 deficiency is associated with poor prognosis of renal cell carcinoma and resistance to sunitinib treatment.

INTRODUCTION: Nuclear envelope spectrin repeat protein (Nesprin) 1 encoded by SYNE1, crucially regulates the morphology and functions of the cell. Mutations in the SYNE1 gene are associated with various diseases; however, their significance in renal cell carcinoma (RCC) remains unknown. In this study, we have investigated the association of SYNE1/Nesprin1 with the progression and prognosis of clear cell RCC (ccRCC). METHODS: In silico analyses of publicly available datasets of patients with RCC were performed. Based on the cohort data, Nesprin1 expression in nephrectomized tissue samples acquired from patients with ccRCC was analyzed using immunohistochemical staining. The invasion, migration, and proliferation of the SYNE1-knockdown human RCC cell lines were analyzed in vitro; moreover, RNA sequencing and Gene Set Enrichment Analysis were conducted to study the molecular mechanism underlying the association of SYNE1/Nesprin1 with prognosis of RCC. RESULTS: Patients with RCC-associated SYNE1 gene mutations exhibited significantly worse overall and progression-free survivals. Patients with Nesprin1-negative ccRCC tumors exhibit significantly poorer overall, cancer-specific, and recurrence-free survival rates than those recorded in the Nesprin1-positive group. SYNE1 knockdown enhanced the invasion and migration of RCC cells, however, it did not influence the proliferation of cells. RNA sequencing and Gene Set Enrichment Analysis revealed that SYNE1 knockdown significantly altered the expression of genes associated with oxidative phosphorylation. Consistently, patients with RCC exhibiting low SYNE1 expression, who were treated with the vascular endothelial growth factor receptor inhibitor sunitinib, had worse progression-free survival. CONCLUSIONS: The results indicate that the expression of SYNE1/Nesprin1 and SYNE1 mutations in patients with RCC are closely linked to their prognosis and responsiveness to sunitinib treatment.

BACH1 promotes clear cell renal cell carcinoma progression by upregulating oxidative stress-related tumorigenicity.

The carcinogenesis and progression of renal cell carcinoma (RCC), a heterogeneous cancer derived from renal tubular epithelial cells, is closely related to oxidative stress responses (OSRs). Oxidative stress responses participate in various biological processes related to the metabolism and metastatic potential of cancer such as inflammation, epithelial-mesenchymal transition (EMT), and angiogenesis. In this study, we investigated the role of broad complex-tramtrack-bric-a-brac and cap 'n' collar homology 1 (BACH1), a key transcription factor for OSRs, in clear cell RCC (ccRCC) development and prognosis. The poor prognosis and elevation of serum inflammation markers in nephrectomized ccRCC patients were correlated with the intratumor expression of BACH1 accompanied by a downregulation of heme oxygenase-1. BACH1 contributes to the invasion and migration abilities of RCC cell lines without affecting their proliferation in vitro. In contrast, BACH1 contributes to tumor progression in vivo, in relation to OSRs with the activation of EMT-related pathways. BACH1 involvement in other OSR-linked pathways, including inflammatory responses, angiogenesis, and mTOR signaling, was further revealed by RNA sequencing analysis of BACH1-knockdown cells. In conclusion, the crucial role of BACH1 in the pathogenesis and poor prognosis of ccRCC through the promotion of OSRs is suggested.

Re-evaluation of over-the-counter histamine H1-receptor antagonists based on their effects on murine models of allergen-induced nasal hyperresponsiveness.

T cells play an essential role in the development of allergen-induced nasal hyperresponsiveness (NHR), a pathophysiological response in allergic rhinitis. The effects of histamine H1-receptor antagonists (antihistamines) on murine NHR models were investigated. Intragastric epinastine, fexofenadine, and loratadine administration suppressed allergen-induced immediate nasal response but not NHR in immunized mice. Regardless of the alleviation of stimulation-induced Th2 cytokine expression by loratadine and desloratadine in vitro, allergen-induced NHR and nasal eosinophil infiltration in Th2 cell-transferred mice were unaffected by loratadine in vivo. This influence on T cell-mediated NHR was excluded from the pharmacological effects of antihistamines.

Paternal knockout of Slc38a4/SNAT4 causes placental hypoplasia associated with intrauterine growth restriction in mice.

The placenta is critical in mammalian embryonic development because the embryo's supply of nutrients, including amino acids, depends solely on mother-to-embryo transport through it. However, the molecular mechanisms underlying this amino acid supply are poorly understood. In this study, we focused on system A amino acid transporters Slc38a1/SNAT1, Slc38a2/SNAT2, and Slc38a4/SNAT4, which carry neutral, short-side-chain amino acids, to determine their involvement in placental or embryonic development. A triple-target CRISPR screen identified Slc38a4/SNAT4 as the critical amino acid transporter for placental development in mice. We established mouse lines from the CRISPR founders with large deletions in Slc38a4 and found that, consistent with the imprinted paternal expression of Slc38a4/SNAT4 in the placenta, paternal knockout (KO) but not maternal KO of Slc38a4/SNAT4 caused placental hypoplasia associated with reduced fetal weight. Immunostaining revealed that SNAT4 was widely expressed in differentiating cytotrophoblasts and maturing trophoblasts at the maternal-fetal interface. A blood metabolome analysis revealed that amino acid concentrations were globally reduced in Slc38a4/SNAT4 mutant embryos. These results indicated that SNAT4-mediated amino acid transport in mice plays a major role in placental and embryonic development. Given that expression of Slc38a4 in the placenta is conserved in other species, our Slc38a4/SNAT4 mutant mice could be a promising model for the analysis of placental defects leading to intrauterine growth restriction in mammals.

Expression and Function of Nicotinic Acetylcholine Receptors in Induced Regulatory T Cells.

A contribution of the cholinergic system to immune cell function has been suggested, though the role of nicotine and its receptors in T cells, especially regulatory T (Treg) cells, is unclear. We herein investigated the expression and function of nicotinic acetylcholine receptors (nAChRs) in murine-induced Treg (iTreg) cells. Upon differentiation of naive BALB/c T cells into iTreg cells and other T-cell subsets, the effect of nicotine on cytokine production and proliferation of iTreg cells was examined. The expression of nAChRs and its regulatory mechanisms were comparatively analyzed among T-cell subsets. Stimulation-induced transforming growth factor-ß1 (TGF-ß1) production of iTreg cells was suppressed by nicotine, whereas interleukin (IL)-10 production and proliferation was not affected. α2-, α5-, α9-, and ß2-nAChRs were differentially expressed in naive, Th1, Th2, Th9, Th17, and iTreg cells. Among these cell types, the α9-nAChR was particularly upregulated in iTreg cells via its gene promoter, but not through tri-methylation at the 4th lysine residue of the histone H3-dependent mechanisms. We conclude that the immunoregulatory role of Treg cells is modified by the cholinergic system, probably through the characteristic expression of nAChRs.

Generation of chimeric mice with spermatozoa fully derived from embryonic stem cells using a triple-target CRISPR method for Nanos3†.

Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo. However, the current cKO method requires considerable time and effort because of the need to generate two gene-modified mouse strains (Cre transgenic and loxP knockin) for crossing. Here, we examined whether we could analyze the germ cell-related functions of embryonic lethal genes in F0 chimeric mice by restricting the origin of germ cells to mutant embryonic stem cells (ESCs). We confirmed that the full ESC origin of spermatozoa in fertile chimeric mice was achieved by the CRISPR/Cas9 system using three guide RNAs targeting Nanos3, which induced germ cell depletion in the host blastocyst-derived tissues. Among these fertile chimeric mice, those from male ESCs with a Dnmt3b mutation, which normally causes embryo death, also produced F1 mice derived exclusively from the mutant ESCs. Thus, our new chimeric strategy readily revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach enables us to analyze the germ cell functions of embryonic lethal genes in the F0 generation without using the current cKO method.

Role of CD4+ T Cells in Allergic Airway Diseases: Learning from Murine Models.

The essential contribution of CD4+ T cells in allergic airway diseases has been demonstrated, especially by using various murine models of antigen-induced airway inflammation. In addition to antigen-immunized mouse models employing mast cell-deficient mice and CD4+ T cell-depleting procedure, antigen-specific CD4+ T cell transfer models have revealed the possible development of allergic inflammation solely dependent on CD4+ T cells. Regardless of the classical Th1/Th2 theory, various helper T cell subsets have the potential to induce different types of allergic inflammation. T cell receptor (TCR)-transgenic (Tg) mice have been used for investigating T cell-mediated immune responses. Besides, we have recently generated cloned mice from antigen-specific CD4+ T cells through somatic cell nuclear transfer. In contrast to TCR-Tg mice that express artificially introduced TCR, the cloned mice express endogenously regulated antigen-specific TCR. Upon antigen exposure, the mite antigen-reactive T cell-cloned mice displayed strong airway inflammation accompanied by bronchial hyperresponsiveness in a short time period. Antigen-specific CD4+ T cell-cloned mice are expected to be useful for investigating the detailed role of CD4+ T cells in various allergic diseases and for evaluating novel anti-allergic drugs.

Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/µl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

CRISPR/Cas9-mediated knock-in of the murine Y chromosomal Sry gene.

Mammalian zygote-mediated genome editing via the clustered regularly interspaced short palindromic repeats/CRISPR-associated endonuclease 9 (CRISPR/Cas9) system is widely used to generate genome-modified animals. This system allows for the production of loss-of-function mutations in various Y chromosome genes, including Sry, in mice. Here, we report the establishment of a CRISPR-Cas9-mediated knock-in line of Flag-tag sequences into the Sry locus at the C-terminal coding end of the Y chromosome (YSry-flag). In the F1 and successive generations, all male pups carrying the YSry-flag chromosome had normal testis differentiation and proper spermatogenesis at maturity, enabling complete fertility and the production of viable offspring. To our knowledge, this study is the first to produce a stable Sry knock-in line at the C-terminal region, highlighting a novel approach for examining the significance of amino acid changes at the naive Sry locus in mammals.

Defects in the first wave of folliculogenesis in mouse XO ovaries.

In mouse ovaries, the first wave of folliculogenesis perinatally starts near the medullary region, which directs the initial round of follicular growth soon after birth. At the same time, cortical primordial follicles start forming in the ovarian surface region, and then some are cyclically recruited for the second and subsequent rounds of follicular growth. Recent studies suggest different dynamics between the first and subsequent waves of follicular growth in postnatal ovaries. However, the phenotypic differences between these phases remain unclear. Here, we show direct evidence that XO female mice, a murine model for Turner Syndrome, lack the first wave of folliculogenesis. Our histopathological analyses of XX and XO littermates revealed a lack of anti-Müllerian hormone (AMH)-positive primary follicles in the XO ovaries by 4 days post partum (dpp). This loss of first follicles was also confirmed by histological bioassay for SRY-dependent SOX9 inducibility, a specific marker for the first follicular granulosa cells. In contrast, cortical primordial follicles formed properly in XO ovaries, and some of them formed primary and secondary follicles in the subcortical region by 7 dpp. They rapidly developed into late antral follicles, showing similarities to XX littermate ovaries by 21 dpp. These results suggest distinct X-monosomy effects between the first and subsequent waves of follicular growth, highlighting the high susceptibility to elimination of XO oocytes in the first wave of mammalian folliculogenesis.

In vivo dynamics of GFRα1-positive spermatogonia stimulated by GDNF signals using a bead transplantation assay.

In mouse testes, spermatogonial stem cells (SSCs), a subpopulation of GFRα1 (GDNF family receptor-α1)-positive spermatogonia, are widely distributed along the convoluted seminiferous tubules. The proliferation and differentiation of the SSCs are regulated in part by local expression of GDNF (glial cell-derived neurotorphic factor), one of major niche factors for SSCs. However, the in vivo dynamics of the GDNF-stimulated GFRα1-positive spermatogonia remains unclear. Here, we developed a simple method for transplanting DiI-labeled and GDNF-soaked beads into the mouse testicular interstitium. By using this method, we examined the dynamics of GFRα1-positive spermatogonia in the tubular walls close to the transplanted GDNF-soaked beads. The bead-derived GDNF signals were able to induce the stratified aggregate formation of GFRα1-positive undifferentiated spermatogonia by day 3 post-transplantation. Each aggregate consisted of tightly compacted Asingle and marginal Apaired-Aaligned GFRα1-positive spermatogonia and was surrounded by Aaligned GFRα1-negative spermatogonia at more advanced stages. These data not only provide in vivo evidence for the inductive roles of GDNF in forming a rapid aggregation of GFRα1-positive spermatogonia but also indicate the usefulness of this in vivo assay system of various growth factors for the stem/progenitor spermatogonia in mammalian spermatogenesis.

Heterogeneity in sexual bipotentiality and plasticity of granulosa cells in developing mouse ovaries.

In mammalian sex determination, SRY directly upregulates the expression of SOX9, the master regulatory transcription factor in Sertoli cell differentiation, leading to testis formation. Without SRY action, the bipotential gonadal cells become pre-granulosa cells, which results in ovarian follicle development. When, where and how pre-granulosa cells are determined to differentiate into developing ovaries, however, remains unclear. By monitoring SRY-dependent SOX9 inducibility (SDSI) in an Sry-inducible mouse system, we were able to identify spatiotemporal changes in the sexual bipotentiality/plasticity of ovarian somatic cells throughout life. The early pre-granulosa cells maintain the SDSI until 11.5 d.p.c., after which most pre-granulosa cells rapidly lose this ability by 12.0 d.p.c. Unexpectedly, we found a subpopulation of the pre-granulosa cells near the mesonephric tissue that continuously retains SDSI throughout fetal and early postnatal stages. After birth, these SDSI-positive pre-granulosa cells contribute to the initial round of folliculogenesis by the secondary follicle stage. In experimental sex reversal of 13.5-d.p.c. ovaries grafted into adult male nude mice, the differentiated granulosa cells re-acquire the SDSI before other signs of masculinization. Our data provide direct evidence of an unexpectedly high sexual heterogeneity of granulosa cells in developing mouse ovaries in a stage- and region-specific manner. Discovery of such sexually bipotential granulosa cells provides a novel entry point to the understanding of masculinization in various cases of XX disorders of sexual development in mammalian ovaries.

Early gonadogenesis in mammals: significance of long and narrow gonadal structure.

In mammalian embryogenesis, the gonadal primordium arises from the thickening of the coelomic epithelium, which results in a pair of extremely long and narrow gonadal structures along the anteroposterior axis. These gonadal structures are conserved in various mammalian species, suggesting a great advantage in properly receiving migrating primordial germ cells (PGCs) that are widely scattered throughout the hindgut tube. Soon after the PGCs settle, the bipotential gonads undergo sex determination into testes or ovaries by the sex-determining gene, Sry, which is expressed in supporting cell precursors in a center-to-pole manner. Such a long, narrow gonadal structure bestows a considerable time lag on Sry expression between the center and pole regions, but testiculogenesis with cord formation and Leydig cell differentiation occurs synchronously throughout the whole organ. This synchronous testiculogenesis could be explained by a positive-feedback mechanism between SOX9 (another SRY-related transcription factor) and FGF9 downstream of Sry. FGF signals are likely secreted from the center region, rapidly diffuse into the poles, and then induce the establishment of SOX9 expression in Sertoli cells in the pole domains. This work focuses on recent knowledge of the molecular and cellular events of PGC migration, gonadogenesis, and testiculogenesis, and their biological significance in mammalian embryogenesis.

α7 nicotinic acetylcholine receptor agonist attenuates allergen-induced immediate nasal response in murine model of allergic rhinitis.

The expression of nicotinic acetylcholine receptor (nAChR) subunits on various immune cells suggests their involvement in allergic rhinitis. However, how exactly they contribute to this pathogenesis is not yet confirmed. Our present study examined the therapeutic potential of GTS-21, an α7 nAChR agonist, for treating allergic rhinitis by employing its mouse models. GTS-21 treatment reduced allergen-induced immediate nasal response in ovalbumin (OVA)-sensitized model. However, nasal hyperresponsiveness or eosinophil infiltration elicited in either the OVA-sensitized or T helper 2 cell-transplanted model was not affected by GTS-21. GTS-21 did not alter allergen-induced passive cutaneous anaphylaxis response in anti-dinitrophenyl IgE-sensitized mice. This evidence implies GTS-21's potential to alleviate allergic rhinitis without perturbing T cells or mast cells.

Evolutionary mechanisms underlying the diversification of nuclear factor of activated T cells across vertebrates.

The mechanisms of immunity linked to biological evolution are crucial for understanding animal morphogenesis, organogenesis, and biodiversity. The nuclear factor of activated T cells (NFAT) family consists of five members (NFATc1-c4, 5) with different functions in the immune system. However, the evolutionary dynamics of NFATs in vertebrates has not been explored. Herein, we investigated the origin and mechanisms underlying the diversification of NFATs by comparing the gene, transcript and protein sequences, and chromosome information. We defined an ancestral origin of NFATs during the bilaterian development, dated approximately 650 million years ago, where NFAT5 and NFATc1-c4 were derived independently. The conserved parallel evolution of NFATs in multiple species was probably attributed to their innate nature. Conversely, frequent gene duplications and chromosomal rearrangements in the recently evolved taxa have suggested their roles in the adaptive immune evolution. A significant correlation was observed between the chromosome rearrangements with gene duplications and the structural fixation changes in vertebrate NFATs, suggesting their role in NFAT diversification. Remarkably, a conserved gene structure around NFAT genes with vertebrate evolutionary-related breaking points indicated the inheritance of NFATs with their neighboring genes as a unit. The close relationship between NFAT diversification and vertebrate immune evolution was suggested.

Generation of reporter mice for detecting the transcriptional activity of nuclear factor of activated T cells.

Nuclear factor of activated T cells (NFAT) is a transcription factor essential for immunological and other biological responses. To develop analyzing system for NFAT activity in vitro and in vivo, we generated reporter mouse lines introduced with NFAT-driven enhanced green fluorescent protein (EGFP) expressing gene construct. Six tandem repeats of -286 to -265 of the human IL2 gene to which NFAT binds in association with its co-transcription factor, activator protein (AP)-1, was conjunct with thymidine kinase minimum promoter and following EGFP coding sequence. Upon introduction of the resulting reporter cassette into C57BL/6 fertilized eggs, the transgenic mice were obtained. Among 7 transgene-positive mice in 110 mice bone, 2 mice showed the designated reporter mouse character. Thus, the EGFP fluorescence of CD4+ and CD8+ T cells in these mice was enhanced by stimulation through CD3 and CD28. Each of phorbol 12-myristate 13-acetate (PMA) and ionomycin (IOM) stimulation weakly but their combined stimulation strongly enhanced EGFP expression. The stimulation-induced EGFP upregulation was also observed following T cell subset differentiation in a different manner. The EGFP induction by PMA + IOM stimulation was more potent than that by CD3/CD28 stimulation in helper T (Th)1, Th2, Th9, and regulatory T cells, while both stimulation conditions displayed the equivalent EGFP induction in Th17 cells. Our NFAT reporter mouse lines are useful for analyzing stimulation-induced transcriptional activation mediated by NFAT in cooperation with AP-1 in T cells.

Intracytoplasmic sperm injection induces transgenerational abnormalities in mice.

In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are 2 major assisted reproductive techniques (ARTs) used widely to treat infertility. Recently, spermatogonial transplantation emerged as a new ART to restore fertility to young patients with cancer after cancer therapy. To examine the influence of germ cell manipulation on behavior of offspring, we produced F1 offspring by a combination of two ARTs, spermatogonial transplantation and ICSI. When these animals were compared with F1 offspring produced by ICSI using fresh wild-type sperm, not only spermatogonial transplantation-ICSI mice but also ICSI-only control mice exhibited behavioral abnormalities, which persisted in the F2 generation. Furthermore, although these F1 offspring appeared normal, F2 offspring produced by IVF using F1 sperm and wild-type oocytes showed various types of congenital abnormalities, including anophthalmia, hydrocephalus, and missing limbs. Therefore, ARTs can induce morphological and functional defects in mice, some of which become evident only after germline transmission.