Biology subjects

Takahashi, G.

Publications and source records attributed to Takahashi, G..

8 recordsLinked to original sources

bioRxiv2026

Initiation of asexual reproduction by the AP2/ERF gene GEMMIFER in Marchantia polymorpha

Plants can propagate their own clones through asexual reproduction. Genetic and hormonal factors regulating asexual reproduction have begun to be elucidated in the liverwort Marchantia polymorpha, which produce asexual propagules called gemmae within the gemma cups. Here, we report an AP2/ERF family gene, GEMMIFER (MpGMFR), as a key regulator of asexual reproduction in M. polymorpha. Suppression of MpGMFR function using genome editing and amiRNA results in the loss of gemma and gemma cup formation. In contrast, activation of MpGMFR function using a dexamethasone inducible system promotes gemma and/or gemma cup formation depending on the induction conditions. Notably, transient strong activation of MpGMFR induced gemma initial cells at the meristem, which develop into mature gemmae capable of reproducing as new individuals after detachment. These results show that MpGMFR is necessary and sufficient for the initiation of asexual reproduction at the meristem. MpGMFR expression was detected from the meristem through to the early stages of gemma development including the gemma cup floor cells. MpGMFR expression precedes the expression of GEMMA CUP-ASSOCIATED MYB1 (MpGCAM1) in early gemma development, supporting the notion that MpGMFR initiates asexual reproduction. We anticipate this finding to be a foundation to study the evolution of extra meristem formation in plant bodies that may have made a significant contribution to the prosperity of plants on land.

plant biology↗

bioRxiv2025

The minimal cell-cycle control system in Marchantia as a framework for understanding plant cell proliferation

The regulation of cell division is broadly conserved across eukaryotes, governed by cyclins and cyclin-dependent kinases (CDKs) to coordinate progression through the cell cycle. Plants have evolved a complex set of cell-cycle genes with unique features. The high number of cyclin-CDK pairs in flowering plants complicates functional studies due to redundancy and diversification. It is critical to study simple systems in other plant lineages to better understand the functional integration of the cell-cycle control machinery and its evolution across land plants. Through a comprehensive phylogenetic analysis, we show that non-seed plants possess a simple repertoire of cyclin and CDK proteins, suggesting that the observed complexity in seed plants is a derived trait. The liverwort Marchantia polymorpha possesses a streamlined set of core cell cycle genes with minimal redundancy during vegetative development. Using single-cell RNA-seq and fluorescent reporters, we found a precise, phase-specific expression pattern for cell cycle genes. We demonstrated in vivo that only three cyclins are active, one at a given phase, without redundancy. Functional analyses revealed that MpCYCD;1 promotes cell cycle re-entry and disrupts differentiation, while overexpression of MpCYCA or MpCYCB;1 arrests the cell cycle, consistent with their respective roles at G1, S, and G2/M progression. Our findings highlight the functional conservation of mechanisms for cell-cycle control across eukaryotes and provide insights into its ancestral state, revealing a minimal set of functional components required for multicellular development. This study advances our understanding of fundamental aspects of cell-cycle regulation and opens new possibilities for engineering plant growth.

plant biology↗

bioRxiv2024

High-throughput robotic isolation of human iPS cell clones reveals frequent homozygous induction of identical genetic manipulations by CRISPR-Cas9

Genome editing in human iPS cells is a powerful approach in regenerative medicine. CRISPR-Cas9 is the most common genome editing tool, but it often induces byproduct insertions and deletions in addition to the desired edits. Therefore, genome editing of iPS cells produces diverse genotypes. Existing assays mostly analyze genome editing results in cell populations, but not in single cells. However, systematic profiling of genome editing outcomes in single iPS cells was lacking. In this study, we developed a method for high-throughput iPS cell clone isolation based on the precise robotic picking of cell clumps derived from single cells grown in extracellular matrices. We analyzed over 1,000 genome-edited iPS cell clones and found that homozygous editing was much more frequent than heterozygous editing. We also observed frequent homozygous induction of identical genetic manipulations, including insertions and deletions. Our new cloning method and findings will facilitate the application of genome editing to human iPS cells.

bioengineering↗

bioRxiv2023

Control of stem cell behavior by CLE-JINGASA signaling in the shoot apical meristem in Marchantia polymorpha

Land plants undergo indeterminate growth by the activity of meristems in both gametophyte (haploid) and sporophyte (diploid) generations1-3. In the sporophyte of the flowering plant Arabidopsis thaliana, the apical meristems are located at the shoot and root tips, in which a number of regulatory gene homologs are shared for their development, implying deep evolutionary origins4-7. However, little is known about their functional conservation with gametophytic meristems in distantly related land plants such as bryophytes8-17, even though genomic studies have revealed the subfamily-level diversity of regulatory genes is mostly conserved throughout land plants18-23. Here we show that a NAM/ATAF/CUC (NAC) domain transcription factor, JINGASA (MpJIN), acts downstream of CLV3/ESR-related (CLE) peptide signaling and controls stem cell behavior in the gametophytic shoot apical meristem of the liverwort Marchantia polymorpha. We found that MpJIN shows heterogeneous expression in space and time within the stem cell zone, associated with the loss and de novo specification of stem cell identity at the branching event. Consistent with the expression pattern, induction of MpJIN results in ectopic periclinal cell division in the stem cell zone and meristem termination. Comparative expression analysis suggests that the function of JIN/FEZ subfamily genes was shared between the shoot apical meristems in the gametophyte and sporophyte generations in early land plants but was lost in certain lineages including the flowering plant A. thaliana.

plant biology↗

bioRxiv2022

Genome Editing Is Induced in a Binary Manner in Single Human Cells

Even when precise nucleotide manipulations are intended, the outcomes of genome editing can be diverse, often including random insertions and deletions. The combinations and frequencies of these different outcomes in single cells are critical not only in the generation of genetically modified cell lines but also in the evaluation of the clinical effects of genome editing therapies. However, current methods only analyze cell populations, not single cells. Here, we utilized the Single Particle isolation System (SPiS) for the efficient isolation of single cells to systematically analyze genome editing results in individual human cultured cells. As a result, we discovered that genome editing induction has a binary nature, that is, the target alleles of cells tend to be all edited or not edited at all. This study enhances our understanding of the induction mechanism of genome editing and provides a new strategy to analyze genome editing outcomes in single cells.

bioengineering↗

bioRxiv2021

Retinoids rescue ceruloplasmin secretion and alleviate oxidative stress in Wilson's disease-specific hepatocytes

Wilsons disease (WD) is a copper metabolic disorder, which is caused by defective ATP7B function. Here, we have generated induced pluripotent stem cells (iPSCs) from WD patients carrying compound heterozygous mutations on ATP7B. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously-corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Transcriptome analysis identified abnormalities of retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Although the expression level of ATP7B protein was variable among WD-specific hepatocytes, the expression and secretion of ceruloplasmin (Cp), which is a downstream copper carrier in plasma, were consistently decreased. Cp secretion-based drug screening identified all-trans retinoic acid (ATRA) as promising candidates for rescuing Cp secretion. ATRA also alleviated reactive oxygen species (ROS) production induced by lipid accumulation in WD-specific hepatocytes. Our patient-derived iPSC-based hepatic models provide potential therapeutics for liver steatosis in WD and other fatty liver diseases.

cell biology↗

bioRxiv2021

An evolutionarily conserved coreceptor gene is essential for CLAVATA signaling in Marchantia polymorpha

Growth and development of land plants are controlled by CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) family of peptide hormones. In contrast to the genetic diversity of CLE family in flowering plants, the liverwort Marchantia polymorpha possesses a minimal set of CLE, MpCLE1(TDIF homolog) and MpCLE2 (CLV3 homolog). MpCLE1 and MpCLE2 peptides exert distinct function at the apical meristem of M. polymorpha gametophyte via specific receptors, MpTDIF RECEPTOR (MpTDR) and MpCLAVATA1 (MpCLV1), respectively, both belonging to the subclass XI of leucine-rich repeat receptor-like kinases (LRR-RLKs). Biochemical and genetic studies in Arabidopsis have shown that TDR/PXY family and CLV1/BAM family recognize the CLE peptide ligand in a heterodimeric complex with a member of subclass-II coreceptors. Here we show that three LRR-RLK genes of M. polymorpha are classified into subclass II, representing three distinct subgroups evolutionarily conserved in land plants. To address the involvement of subclass-II coreceptors in M. polymorpha CLE signaling, we performed molecular genetic analysis on one of them, MpCLAVATA3 INSENSITIVE RECEPTOR KINASE (MpCIK). Two knockout alleles for MpCIK formed narrow apical meristems marked by promMpYUC2:GUS marker, which were not expanded by MpCLE2 peptide treatment, phenocopying Mpclv1. Loss of sensitivity to MpCLE2 peptide was also observed in gemma cup formation in both Mpclv1 and Mpcik. Biochemical analysis using a Nicotiana benthamiana transient expression system revealed weak association between MpCIK and MpCLV1, as well as MpCIK and MpTDR. While MpCIK may also participate in MpCLE1 signaling, our data show that the conserved CLV3-CLV1-CIK module functions in M. polymorpha, controlling meristem activity for development and organ formation for asexual reproduction.

plant biology↗

bioRxiv2020

Rapid, precise quantification of large DNA excisions and inversions by ddPCR

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (>20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

molecular biology↗