Takafumi Sakai, Masahiro Kawakita, Arashi Seki, Katsuyoshi Takaoka, Misaki Kurata, Junpei Torikai, Sohshiroh Atsumi, Kanji Yawata, Kentaro Noi, Yosuke Fukutani, Keiichi Noguchi, Hirofumi Kiyokawa, Mitsuru Morimoto, Eri Takeuchi, Katsura Minegishi, Yoshitsugu Aoki, Shoko Fujimura, Takayuki Nishizaka, Takanari Inoue, Kentaro Nagaoka, Hiroshi Tsugawa, Hisayoshi Hayashi, Hiroshi Ishiguro, Takuma Kozono, Ikuroh Ohsawa, Yasunori Fujita, Hiroaki Matsunami, Hiroshi Hamada and Kyosuke Shinohara : Tppp3 determines basal body positioning and identity of respiratory cilia via microtubule assembly and sphingolipid homeostasis, Proceedings of the National Academy of Sciences of the United States of America, 122, 49, e2503931122, 2025.
(要約)
Cilia are hair-like organelles that protrude from the cell surface. In mammals, tracheal multiciliated cells (MCCs) play an important role in elimination of hazardous microorganisms by driving a unidirectional mucus flow. Although uniform orientation of ciliary beating is critical for the unidirectional flow, it remains unknown how MCCs establish uniform orientations and maintain identities of hundreds of ciliary membranes. This study focuses on investigating the roles of Tubulin Polymerization Promoting Family Member 3 (Tppp3) in MCC function. We generated a Tppp3-deficient mouse (Tppp3∆ex2-4/∆ex2-4; Tppp3 knockout (KO)) and found that the Tppp3 KO mouse exhibited cough and hyposmia phenotype. The loss of Tppp3 disrupted the apical microtubules (MTs) meshwork in the tracheal MCCs, leading to random orientation and alignment of basal bodies (BBs) of the motile cilia. Unexpectedly, aberrant ciliary membrane fusions occurred in the trachea of the Tppp3 KO mice. We examined the underlying molecular mechanism of the ciliary membrane fusion by isolating the tracheal cilium. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis as well as pharmacological analysis revealed that hyperaccumulation of a long chain ceramide at the ciliary membrane caused the membrane fusion. In addition, sensory cilia formation was impaired in the olfactory sensory neuron of the Tppp3 KO mice. Due to the lack of Tppp3, dendritic MT assembly that underlies long-range migration of BBs toward the cell surface was impaired. These findings demonstrate that Tppp3, as well as the defined intracellular MT architecture, regulate proper orientation/subcellular positioning of BBs and the independency of individual motile cilium membranes.
Embryonic diapause is a temporary suspension of proliferation in mammalian pre-implantation embryos, allowing for reactivation later. Cells in mouse diapause embryos enter the G0 phase within 7 days of diapause initiation. Here, we show that approximately 5% of cells in embryonic tissues continue to proliferate even after 7 days of diapause. Transcriptome and phenotypic analyses reveal that p53 facilitated DNA damage repair via p21-mediated cell cycle arrest and regulated epiblast cell numbers via Bax-mediated apoptosis. Moreover, epiblast cell numbers strongly correlated with reactivation rates, with deviations from optimal levels impairing successful reactivation. Our findings call into question the conventional view that all cells in diapause embryos uniformly enter G0. We established epiblast characteristics as predictive factors for determining reactivation success, the defining event of diapause.
The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.
Katarina Harasimov, L. Rebecca Gorry, M. Luisa Welp, Mae Sarah Penir, Yehor Horokhovskyi, Shiya Cheng, Katsuyoshi Takaoka, Alexandra Stützer, Sophie Ann Frombach, Lisa Tavares Ana Taylor, Monika Raabe, Sara Haag, Debojit Saha, Katharina Grewe, Vera Schipper, O. Silvio Rizzoli, Henning Urlaub, Juliane Liepe and Melina Schuh : The maintenance of oocytes in the mammalian ovary involves extreme protein longevity, Nature Cell Biology, 26, 7, 1124-1138, 2024.
(要約)
Women are born with all of their oocytes. The oocyte proteome must be maintained with minimal damage throughout the woman's reproductive life, and hence for decades. Here we report that oocyte and ovarian proteostasis involves extreme protein longevity. Mouse ovaries had more extremely long-lived proteins than other tissues, including brain. These long-lived proteins had diverse functions, including in mitochondria, the cytoskeleton, chromatin and proteostasis. The stable proteins resided not only in oocytes but also in long-lived ovarian somatic cells. Our data suggest that mammals increase protein longevity and enhance proteostasis by chaperones and cellular antioxidants to maintain the female germline for long periods. Indeed, protein aggregation in oocytes did not increase with age and proteasome activity did not decay. However, increasing protein longevity cannot fully block female germline senescence. Large-scale proteome profiling of ~8,890 proteins revealed a decline in many long-lived proteins of the proteostasis network in the aging ovary, accompanied by massive proteome remodeling, which eventually leads to female fertility decline.
Yumiko Hayashi, Masakazu Hashimoto, Katsuyoshi Takaoka, Tatsuya Takemoto, Nobuyuki Takakura and Hiroyasu Kidoya : Tumor endothelial cell-derived Sfrp1 supports the maintenance of cancer stem cells via Wnt signaling, In Vitro Cellular & Developmental Biology. Animal, 60, 10, 1123-1131, 2024.
(要約)
Cancer stem cells (CSCs), which are critical targets for cancer therapy as they are involved in drug resistance to anticancer drugs, and metastasis, are maintained by angiocrine factors produced by particular niches that form within tumor tissue. Secreted frizzled-related protein 1 (Sfrp1) is an extracellular protein that modulates Wnt signaling. However, the cells that produce Sfrp1 in the tumor environment and its function remain unclear. We aimed to elucidate angiocrine factors related to CSC maintenance, focusing on Sfrp1. Although Sfrp1 is a Wnt pathway-related factor, its impact on tumor tissues remains unknown. We investigated the localization of Sfrp1 in tumors and found that it is expressed in some tumor vessels. Analysis of mice lacking Sfrp1 showed that tumor growth was suppressed in Sfrp1-deficient tumor tissues. Flow cytometry analysis indicated that CSCs were maintained in the early tumor growth phase in the Sfrp1 knockout (KO) mouse model of tumor-bearing cancer. However, tumor growth was inhibited in the late tumor growth phase because of the inability to maintain CSCs. Real-time PCR results from tumors of Sfrp1 KO mice showed that the expression of Wnt signaling target genes significantly decreased in the late stage of tumor growth. This suggests that Sfrp1, an angiocrine factor produced by the tumor vascular niche, is involved in Wnt signaling-mediated mechanisms in tumor tissues.
Sylvain Hiver, Natsumi Shimizu-Mizuno, Yayoi Ikawa, Eriko Kajikawa, Xiaorei Sai, Hiromi Nishimura, Katsuyoshi Takaoka, Osamu Nishimura, Shigehiro Kuraku, Satoshi Tanaka and Hiroshi Hamada : Gse1, a component of the CoREST complex, is required for placenta development in the mouse, Developmental Biology, 498, 97-105, 2023.
(要約)
Gse1 is a component of the CoREST complex that acts as an H3K4 and H3K9 demethylase and regulates gene expression. Here, we examined the expression and role of Gse1 in mouse development. Gse1 is expressed in male and female germ cells and plays both maternal and zygotic roles. Thus, maternal deletion of Gse1 results in a high incidence of prenatal death, and zygotic deletion leads to embryonic lethality from embryonic day 12.5 (E12.5) and perinatal death. Gse1 is expressed in the junctional zone and the labyrinth of the developing placenta. Gse1 mutant (Gse1) placenta begins to exhibit histological defects from E14.5, being deficient in MCT4 syncytiotrophoblast II. The number of various cell types was largely maintained in the mutant placenta at E10.5, but several genes were upregulated in giant trophoblasts at E10.5. Placenta-specific deletion of Gse1 with Tat-Cre suggested that defects in Gse1 embryos are due to placental function deficiency. These results suggest that Gse1 is required for placental development in mice, and in turn, is essential for embryonic development.
Xiaorei Sai, Yayoi Ikawa, Hiromi Nishimura, Katsutoshi Mizuno, Eriko Kajikawa, A Takanobu Katoh, Toshiya Kimura, Hidetaka Shiratori, Katsuyoshi Takaoka, Hiroshi Hamada and Katsura Minegishi : Planar cell polarity-dependent asymmetric organization of microtubules for polarized positioning of the basal body in node cells., Development, 149, 9, 2022.
(要約)
For left-right symmetry breaking in the mouse embryo, the basal body must become positioned at the posterior side of node cells, but the precise mechanism for this has remained unknown. Here, we examined the role of microtubules (MTs) and actomyosin in this basal body positioning. Exposure of mouse embryos to agents that stabilize or destabilize MTs or F-actin impaired such positioning. Active myosin II was detected at the anterior side of node cells before the posterior shift of the basal body, and this asymmetric activation was lost in Prickle and dachsous mutant embryos. The organization of basal-body associated MTs (baMTs) was asymmetric between the anterior and posterior sides of node cells, with anterior baMTs extending horizontally and posterior baMTs extending vertically. This asymmetry became evident after polarization of the PCP core protein Vangl1 and before the posterior positioning of the basal body, and it also required the PCP core proteins Prickle and dachsous. Our results suggest that the asymmetry in baMT organization may play a role in correct positioning of the basal body for left-right symmetry breaking.
W Gerard Dougherty, Katsutoshi Mizuno, Tabea Nöthe-Menchen, Yayoi Ikawa, Karsten Boldt, Asaf Ta-Shma, Isabella Aprea, Katsura Minegishi, Yuan-Ping Pang, Petra Pennekamp, T Niki Loges, Johanna Raidt, Rim Hjeij, Julia Wallmeier, Huda Mussaffi, Zeev Perles, Orly Elpeleg, Franziska Rabert, Hidetaka Shiratori, J Stef Letteboer, Nicola Horn, Samuel Young, Timo Strünker, Friederike Stumme, Claudius Werner, Heike Olbrich, Katsuyoshi Takaoka, Takahiro Ide, Kyaw Wang Twan, Luisa Biebach, Jörg Große-Onnebrink, A Judith Klinkenbusch, Kavita Praveen, C Diana Bracht, M Inga Höben, Katrin Junger, Jana Gützlaff, Sandra Cindrić, Micha Aviram, Thomas Kaiser, Yasin Memari, P Petras Dzeja, Bernd Dworniczak, Marius Ueffing, Ronald Roepman, Kerstin Bartscherer, Nicholas Katsanis, E Erica Davis, Israel Amirav, Hiroshi Hamada and Heymut Omran : CFAP45 deficiency causes situs abnormalities and asthenospermia by disrupting an axonemal adenine nucleotide homeostasis module., Nature Communications, 11, 1, 2020.
(要約)
mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.
(キーワード)
Adenine Nucleotides / Adolescent / Adult / Animals / Asthenozoospermia / Axoneme / CRISPR-Cas Systems / Cilia / Cytoskeletal Proteins / DNA Mutational Analysis / Disease Models, Animal / Epididymis / Female / Flagella / Humans / Loss of Function Mutation / Male / Mice / Mice, Knockout / Middle Aged / Planarians / Respiratory Mucosa / Situs Inversus / Sperm Motility / Tomography, X-Ray Computed / Whole Exome Sequencing
Nobuyuki Kawamura, Katsuyoshi Takaoka, Hiroshi Hamada, Anna-Katerina Hadjantonakis, Ge-Hong Sun-Wada and Yoh Wada : Rab7-Mediated Endocytosis Establishes Patterning of Wnt Activity through Inactivation of Dkk Antagonism, Cell Reports, 31, 10, 107733, 2020.
(要約)
Endocytosis has been proposed to modulate cell signaling activities. However, the role of endocytosis in embryogenesis, which requires coordination of multiple signaling inputs, has remained less understood. We previously showed that mouse embryos lacking a small guanosine triphosphate (GTP)-binding protein Rab7 implicated in endocytic flow are defective in gastrulation. Here, we investigate how subcellular defects associated with Rab7 deficiency are related to the observed developmental defects. Rab7-deficient embryos fail to organize mesodermal tissues due to defects in Wnt-β-catenin signaling. Visceral endoderm (VE)-specific ablation of Rab7 results in patterning defects similar to systemic Rab7 deletion. Rab7 mutants accumulate the Wnt antagonist Dkk1 in the extracellular space and in intracellular compartments throughout the VE epithelium. These data indicate that Rab7-dependent endocytosis regulates the concentration and availability of extracellular Dkk1, thereby relieving the epiblast of antagonism. This intercellular mechanism therefore organizes distinct spatiotemporal patterns of canonical Wnt activity during the peri-gastrulation stages of embryonic development.
Katsuyoshi Takaoka, Hiromi Nishimura and Hiroshi Hamada : Both Nodal signalling and stochasticity select for prospective distal visceral endoderm in mouse embryos, Nature Communications, 8, 1, 1492, 2017.
(要約)
Anterior-posterior (A-P) polarity of mouse embryos is established by distal visceral endoderm (DVE) at embryonic day (E) 5.5. Lefty1 is expressed first at E3.5 in a subset of epiblast progenitor cells (L1epi cells) and then in a subset of primitive endoderm cells (L1dve cells) fated to become DVE. Here we studied how prospective DVE cells are selected. Lefty1 expression in L1epi and L1dve cells depends on Nodal signaling. A cell that experiences the highest level of Nodal signaling begins to express Lefty1 and becomes an L1epi cell. Deletion of Lefty1 alone or together with Lefty2 increased the number of prospective DVE cells. Ablation of L1epi or L1dve cells triggered Lefty1 expression in a subset of remaining cells. Our results suggest that selection of prospective DVE cells is both random and regulated, and that a fixed prepattern for the A-P axis does not exist before the blastocyst stage.
Katsutoshi Mizuno, Kei Shiozawa, Takanobu A Katoh, Katsura Minegishi, Takahiro Ide, Yayoi Ikawa, Hiromi Nishimura, Katsuyoshi Takaoka, Takeshi Itabashi, Atsuko H Iwane, Junichi Nakai, Hidetaka Shiratori and Hiroshi Hamada : Role of Ca2+ transients at the node of the mouse embryo in breaking of left-right symmetry, Science Advances, 6, 30, 1195, 2020.
(要約)
Immotile cilia sense extracellular signals such as fluid flow, but whether Ca plays a role in flow sensing has been unclear. Here, we examined the role of ciliary Ca in the flow sensing that initiates the breaking of left-right (L-R) symmetry in the mouse embryo. Intraciliary and cytoplasmic Ca transients were detected in the crown cells at the node. These Ca transients showed L-R asymmetry, which was lost in the absence of fluid flow or the PKD2 channel. Further characterization allowed classification of the Ca transients into two types: cilium-derived, L-R-asymmetric transients (type 1) and cilium-independent transients without an L-R bias (type 2). Type 1 intraciliary transients occurred preferentially at the left posterior region of the node, where L-R symmetry breaking takes place. Suppression of intraciliary Ca transients delayed L-R symmetry breaking. Our results implicate cilium-derived Ca transients in crown cells in initiation of L-R symmetry breaking in the mouse embryo.