Biology subjects

Aizawa, E.

Publications and source records attributed to Aizawa, E..

7 recordsLinked to original sources

bioRxiv2026

Inflammasome activation drives gasdermin-independent plasma membrane rupture by clustering ninjurin-1 in macrophages

Inflammasome assembly rapidly triggers caspase-1 activation to initiate pyroptosis, an inflammatory cell death characterized by the release of cytosolic contents, including interleukin (IL)-1{beta}/. Here, we report that inflammasome activation drives necrotic cell death independent of gasdermin D (GSDMD) and GSDME, which are essential executors of pyroptosis by forming a pore on the plasma membrane and increasing membrane permeability. NLRP3 inflammasome activation induced necrotic cell death, coupled with IL-1{beta}/ release in Gsdmd-/-Gsdme-/- macrophages. Mechanistically, the oligomerization of ninjurin-1 (NINJ1) was caused by inflammasome activation even in the absence of GSDMD and GSDME. Concordantly, glycine, an inhibitor of NINJ1, blocked plasma membrane permeabilization triggered by inflammasome activation in Gsdmd-/-Gsdme-/- macrophages, but not in WT macrophages. The dimerizer-mediated ASC oligomerization promoted NINJ1-mNeonGreen cluster formation in the absence of GSDMD and GSDME. Moreover, NINJ1 deficiency prevented membrane permeabilization initiated by ASC oligomerization in Gsdmd-/-Gsdme-/- immortalized bone marrow-derived macrophages (iBMDM). Blocking of phosphatidylserine (PtdSer) exposure, a feature of inflammasome-driven necrotic cell death, by Xkr8 deficiency inhibited plasma membrane permeabilization in Gsdmd-/-Gsdme-/- iBMDM. These results suggest that inflammasome-triggered activation of caspase-1 itself drives inflammatory necrotic cell death independent of gasdermins.

cell biology↗

bioRxiv2025

Ex vivo live imaging unveils the dynamics of oocyte formation in mice

Oocyte formation in mammals is a tightly regulated process essential for female fertility, yet the underlying mechanisms remain poorly understood. In this study, we establish an ex vivo culture system that faithfully recapitulates in vivo development and enables long-term live imaging of mouse fetal ovaries. Using high resolution imaging, we capture the dynamic behaviors of germ cells during the development from oogonia to nascent oocytes. We identify pronounced blebbing activity during the mitosis-to-meiosis transition. This behavior is regulated by meiotic initiation signals, underscoring its potential developmental relevance, although its precise role remains unclear. A prevailing model suggests that oocyte formation involves organelle transfer from neighboring germ cells during cyst breakdown. However, through photoconversion-based tracking, we observe no detectable transfer of mitochondria or centrosomes, as organelles remain confined to individual cells. These findings point to alternative mechanisms for cytoplasmic enrichment in oocytes. Our study provides new insights into mammalian oocyte formation and establishes a powerful platform for analyzing germ cell dynamics in real time.

developmental biology↗

bioRxiv2024

Optimization of an adeno-associated viral vector for keratinocytes in vitro and in vivo

BackgroundLocal gene therapies, including in vivo genome editing, are highly anticipated for the treatment of genetic diseases in skin, especially the epidermis. While the adeno-associated virus (AAV) is a potent vector for in vivo gene delivery, the lack of efficient gene delivery methods has limited its clinical applications. ObjectiveTo optimize the AAV gene delivery system with higher gene delivery efficiency and specificity for epidermis and keratinocytes (KCs), using AAV capsid and promoter engineering technologies. MethodsAAV variants with mutations in residues reported to be critical to determine the tropism of AAV2 for KCs were generated by site-directed mutagenesis of AAVDJ. The infection efficiency and specificity for KCs of these variants were compared with those of previously reported AAVs considered to be suitable for gene delivery to KCs in vitro and in vivo. Additionally, we generated an epidermis-specific promoter using the most recent short-core promoter and compared its specificity with existing promoters. ResultsA novel AAVDJ variant capsid termed AAVDJK2 was superior to the existing AAVs in terms of gene transduction efficiency and specificity for epidermis and KCs in vitro and in vivo. A novel tissue-specific promoter, termed the K14 SCP3 promoter, was superior to the existing promoters in terms of gene transduction efficiency and specificity for KCs. ConclusionThe combination of the AAVDJK2 capsid and K14 SCP3 promoter improves gene delivery to epidermis in vivo and KCs in vitro. The novel AAV system may benefit experimental research and development of new epidermis-targeted gene therapies.

bioengineering↗

bioRxiv2023

Comprehensive comparison of female germ cell development in vitro and in vivo identifies epigenetic gene regulation crucial for oocyte development and embryonic competence

Germ cells are the origin of new individuals. Hence, specifying germ cell identity is crucial for reproduction. The recent establishment of in vitro culture systems for generating oocytes from mouse pluripotent stem cells provides a basis for progress in studies of oogenesis and reproductive technology. However, currently the developmental competence of in vitro generated oocytes is low compared to in vivo grown oocytes. The causes underlying poor oocyte quality remain to be determined. By reconstituting germ cell development in culture from different developmental starting points within gametogenesis, we show that the differentiation of primordial germ cells (PGCs) and primordial germ cell-like cells (PGCLCs) to growing oocytes (GROs), as well as the subsequent growth of follicles are critical culture steps for specifying competence of fully-grown oocytes (FGOs) for preimplantation development. A systematic comparison of transcriptomes of single oocytes having undergone different in vitro culture trajectories identifies genes normally upregulated during oocyte growth to be susceptible for mis-regulation during in vitro oogenesis. Many of such genes have been described as targets of Polycomb repressive complexes (PRCs). Deregulation of Polycomb repression therefore likely perturbs the accumulation of cytoplasmic factors and/or setting of chromatin states in FGOs that are required for embryonic development after fertilization. Conversely, in vitro derived oocytes often displayed failure of zygotic genome activation (ZGA) and abnormal acquisition of 5-hydroxymethylcytosine (5hmC) on maternal chromosomes after activation. In addition, subcellular delocalization of pyruvate dehydrogenase (PDH) and of STELLA were observed suggesting new molecular markers for defective oocyte development. Our study identifies epigenetic regulation at an early stage of oogenesis as crucial for developmental competence and suggests specific in vitro culture steps as targets for improving oocyte quality. HighlightsO_LISingle cell transcriptomics and functional assessment of oocyte development from pluripotent stem cells in culture in a stage-specific manner provides a comprehensive resource for comparisons to oogenesis in vivo. C_LIO_LICulture steps for growth and differentiation of reconstituted follicles are critical for defining embryonic competence of in vitro generated oocytes. C_LIO_LIZygotic genome activation failure and epigenetic impairment are hallmarks of in vitro-generated oocytes that fail to develop after activation or fertilization. C_LIO_LIComputational analysis of gene expression changes and chromatin modification patterns identifies specific gene sets that indicate that Polycomb mediated repression is vulnerable during in vitro folliculogenesis. C_LI

developmental biology↗

bioRxiv2021

Cryo-sensitive aggregation triggers NLRP3 inflammasome assemblyin cryopyrin-associated periodic syndrome

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3, which was originally identified as cryopyrin. Familial cold autoinflammatory syndrome (FCAS), the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1{beta}. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1{beta} release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was indispensable for mutated NLRP3-mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation.

immunology↗

bioRxiv2020

Haploid mouse germ cell precursors from embryonic stem cells reveal Xist activation from a single X chromosome

Mammalian haploid cells have applications for genetic screening and substituting gametic genomes. Here we characterize a culture system for obtaining haploid primordial germ cell-like cells (PCGLCs) from haploid mouse embryonic stem cells (ESCs). We find that a haploid genome is maintained in PGCLCs with a high frequency indicating a substantially lower rate of diploidization than somatic cells. Characterization of the differentiating haploid ESCs reveals that Xist is activated from the single X chromosome. This observation suggests that X chromosome inactivation is initiated in haploid cells consistent with a model where autosomal blocking factors set a threshold for X-linked activators. The germline segregates from the epiblast and differs from somatic lineages in gene expression and epigenetic mechanisms. The ability of primordial germ cells for repressing Xist might contribute to the maintenance of a haploid genome.

developmental biology↗

bioRxiv2020

Polyploidy of semi-cloned embryos generated from parthenogenetic haploid embryonic stem cells

In mammals, the fusion of two gametes, an oocyte and a spermatozoon, during fertilization forms a totipotent zygote. There has been no reported case of natural parthenogenesis, in which embryos develop from unfertilized oocytes. The genome and epigenetic information of haploid gametes are crucial for the proper development of embryos. Haploid embryonic stem cells (haESCs) are unique stem cells established from uniparental blastocysts and possess only one set of chromosomes. Previous studies have shown that sperm or oocyte genome can be replaced by haESCs with or without manipulation of genomic imprinting for generation of mice. Recently, these remarkable semi-cloning methods have been applied for screening of key factors of mouse embryonic development. While haESCs have been applied as substitute of gametic genome, the fundamental mechanism how haESCs contribute to the genome of totipotent embryos is unclear. Here, we show the generation of fertile semi-cloned mice by injection of parthenogenetic haESCs (phaESCs) into oocytes after deletions of two differentially methylated regions (DMRs), the IG-DMR and H19-DMR. For characterizing the genome of semi-cloned embryos further we establish ESC lines from semi-cloned blastocysts. We report that polyploid karyotypes are observed frequently in semi-cloned ESCs (scESCs). Our results confirm that mitotically arrested phaESCs provide high efficiency for semi-cloning when the IG-DMR and H19-DMR are deleted. In addition, we highlight the occurrence of polyploidy that needs to be considered for further improvement for development of semi-cloned embryos derived by haESC injection.

developmental biology↗