CVClient

Yoshihiko Umesono

  (梅園 良彦)

Profile Information

Affiliation
University of Hyogo
Degree
博士(理学)(神戸大学)

J-GLOBAL ID
202001002757742836
researchmap Member ID
R000001117

Papers

 41
  • Makoto Mochii, Kai Akizuki, Hero Ossaka, Norie Kagawa, Yoshihiko Umesono, Ken-Ichi T Suzuki
    Developmental biology, 506 42-51, Dec 3, 2023  
    Xenopus laevis is a widely used model organism in developmental and regeneration studies. Despite several reports regarding targeted integration techniques in Xenopus, there is still room for improvement of them, especially in creating reporter lines that rely on endogenous regulatory enhancers/promoters. We developed a CRISPR-Cas9-based simple method to efficiently introduce a fluorescent protein gene into 5' untranslated regions (5'UTRs) of target genes in Xenopus laevis. A donor plasmid DNA encoding an enhanced green fluorescent protein (eGFP) flanked by a genomic fragment ranging from 66 bp to 878 bp including target 5'UTR was co-injected into fertilized eggs with a single guide RNA and Cas9 protein. Injections for krt12.2.L, myod1.S, sox2.L or brevican.S resulted in embryos expressing eGFP fluorescence in a tissue-specific manner, recapitulating endogenous expression of target genes. Integrations of the donor DNA into the target regions were examined by genotyping PCR for the eGFP-expressing embryos. The rate of embryos expressing the specific eGFP varied from 2.1% to 13.2% depending on the target locus and length of the genomic fragment in the donor plasmids. Germline transmission of an integrated DNA was observed. This simple method provides a powerful tool for exploring gene expression and function in developmental and regeneration research in X. laevis.
  • Yuki Shibata, Miyuki Suzuki, Nao Hirose, Ayuko Takayama, Chiaki Sanbo, Takeshi Inoue, Yoshihiko Umesono, Kiyokazu Agata, Naoto Ueno, Ken-ichi T. Suzuki, Makoto Mochii
    Developmental Biology, 489 76-83, Jun, 2022  Peer-reviewed
    Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.
  • Yuki Sato, Yoshihiko Umesono, Yoshihito Kuroki, Kiyokazu Agata, Chikara Hashimoto
    Developmental Biology, 482 55-66, Feb, 2022  Peer-reviewed
    The coincidence of cell cycle exit and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since they may possess a simple cell cycle regulation in which Cdh1 is one of the factors responsible for exiting the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the knockdown of cdh1 caused pronounced accumulation of the stem cells, the progenitor and differentiated cells were decreased. Further analyses indicated that the stem cells with cdh1 knockdown did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle exit. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation.
  • Hayoung Lee, Kanon Hikasa, Yoshihiko Umesono, Tetsutaro Hayashi, Kiyokazu Agata, Norito Shibata
    Development (Cambridge, England), 149(3), Feb 1, 2022  Peer-reviewed
    The regenerative ability of planarians relies on their adult pluripotent stem cell population. Although all stem cells express a piwi homolog, recently it has become possible to classify the piwi+ stem cell population into specialized subpopulations according to the expression of genes related to differentiation. However, piwi+ stem cells behave practically as a homogeneous population after amputation, during which stem cells show accelerated proliferation, named 'induced hyperproliferation'. Here, we show that plac8-A was expressed in almost all of the stem cells, and that a decrease of the plac8-A expression level led to induced hyperproliferation uniformly in a broad stem cell subpopulation after amputation. This reduction of plac8-A expression was caused by activated JNK signaling after amputation. Pharmacological inhibition of JNK signaling caused failure to induce hyperproliferation and resulted in regenerative defects. Such defects were abrogated by simultaneous knockdown of plac8-A expression. Thus, JNK-dependent suppression of plac8-A expression is indispensable for stem cell dynamics involved in regeneration. These findings suggest that plac8-A acts as a molecular switch of piwi+ stem cells for entry into the regenerative state after amputation.
  • Mohammad Abdul Auwal, Makoto Kashima, Osamu Nishimura, Kazutaka Hosoda, Minako Motoishi, Akifumi Kamimura, Akinori Okumura, Kiyokazu Agata, Yoshihiko Umesono
    Development, growth & differentiation, 62(9) 527-539, Dec, 2020  Peer-reviewed
    Planarians belong to the phylum Platyhelminthes and can regenerate their missing body parts after injury via activation of somatic pluripotent stem cells called neoblasts. Previous studies suggested that fibroblast growth factor (FGF) signaling plays a crucial role in the regulation of head tissue differentiation during planarian regeneration. To date, however, no FGF homologues in the Platyhelminthes have been reported. Here, we used a planarian Dugesia japonica model and identified an fgf gene termed Djfgf, which encodes a putative secreted protein with a core FGF domain characteristic of the FGF8/17/18 subfamily in bilaterians. Using Xenopus embryos, we found that DjFGF has FGF activity as assayed by Xbra induction. We next examined Djfgf expression in non-regenerating intact and regenerating planarians. In intact planarians, Djfgf was expressed in the auricles in the head and the pharynx. In the early process of regeneration, Djfgf was transiently expressed in a subset of differentiated cells around wounds. Notably, Djfgf expression was highly induced in the process of head regeneration when compared to that in the tail regeneration. Furthermore, assays of head regeneration from tail fragments revealed that combinatorial actions of the anterior extracellular signal-regulated kinase (ERK) and posterior Wnt/ß-catenin signaling restricted Djfgf expression to a certain anterior body part. This is the region where neoblasts undergo active proliferation to give rise to their differentiating progeny in response to wounding. The data suggest the possibility that DjFGF may act as an anterior counterpart of posteriorly localized Wnt molecules and trigger neoblast responses involved in planarian head regeneration.

Misc.

 62
  • Susanna Fraguas, Yoshihiko Umesono, Kiyokazu Agata, Francesc Cebrià
    Methods in molecular biology (Clifton, N.J.), 1487 303-315, 2017  
    Planarians are an ideal model in which to study stem cell-based regeneration. After amputation, planarian pluripotent stem cells surrounding the wound proliferate to produce the regenerative blastema, in which they differentiate into the missing tissues and structures. Recent independent studies in planarians have shown that Smed-egfr-3, a gene encoding a homologue of epidermal growth factor (EGF) receptors, and DjerkA, which encodes an extracellular signal-regulated kinase (ERK), may control cell differentiation and blastema growth. However, because these studies were carried in two different planarian species, the relationship between these two genes remains unclear. We have optimized anti-pERK immunostaining in Schmidtea mediterranea using the original protocol developed in Dugesia japonica. Both protocols are reported here as most laboratories worldwide work with one of these two species. Using this protocol we have determined that Smed-egfr-3 appears to be necessary for pERK activation during planarian regeneration.
  • 柳原由実, 中川晴香, 前澤孝信, 梅園良彦, 阿形清和, 小林一也
    日本動物学会大会予稿集, 85th, 2014  
  • 細田和孝, 阿形清和, 梅園良彦
    日本動物学会大会予稿集, 85th, 2014  
  • 西村理, 細田和孝, 川口恵里, 矢澤重信, 矢澤重信, 林哲太郎, 林哲太郎, 井上武, 梅園良彦, 梅園良彦, 阿形清和
    日本分子生物学会年会プログラム・要旨集(Web), 37th, 2014  
  • 西村唯, 川口恵里, 矢澤重信, 西村理, 井上武, 阿形清和, 梅園良彦
    日本動物学会大会予稿集, 84th 115, Aug 12, 2013  

Books and Other Publications

 1

Research Projects

 4