記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：{eLife} Sciences Publications, Ltd  

Cellular polarization is fundamental for various biological processes. The Par network system is conserved for cellular polarization. Its core complex consists of Par3, Par6, and aPKC. However, the general dynamic processes that occur during polarization are not well understood. Here, we reconstructed Par-dependent polarity using non-polarized Drosophila S2 cells expressing all three components endogenously in the cytoplasm. The results indicated that elevated Par3 expression induces cortical localization of the Par-complex at the interphase. Its asymmetric distribution goes through three steps: emergence of cortical dots, development of island-like structures with dynamic amorphous shapes, repeating fusion and fission, and polarized clustering of the islands. Our findings also showed that these islands contain a meshwork of unit-like segments. Furthermore, Par-complex patches resembling Par-islands exist in Drosophila mitotic neuroblasts. Thus, this reconstruction system provides an experimental paradigm to study features of the assembly process and structure of Par-dependent cell-autonomous polarity.

DOI： 10.7554/eLife.45559

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Genome-editing technology has revolutionized the field of biology. Here, we report a novel de novo gene-targeting method mediated by in utero electroporation into the developing mammalian brain. Electroporation of donor DNA with the CRISPR/Cas9 system vectors successfully leads to knock-in of the donor sequence, such as EGFP, to the target site via the homology-directed repair mechanism. We developed a targeting vector system optimized to prevent anomalous leaky expression of the donor gene from the plasmid, which otherwise often occurs depending on the donor sequence. The knock-in efficiency of the electroporated progenitors reached up to 40% in the early stage and 20% in the late stage of the developing mouse brain. Furthermore, we inserted different fluorescent markers into the target gene in each homologous chromosome, successfully distinguishing homozygous knock-in cells by color. We also applied this de novo gene targeting to the ferret model for the study of complex mammalian brains. Our results demonstrate that this technique is widely applicable for monitoring gene expression, visualizing protein localization, lineage analysis and gene knockout, all at the single-cell level, in developmental tissues.

DOI： 10.1242/dev.136325

Web of Science

PubMed

researchmap

担当区分：筆頭著者   記述言語：英語   掲載種別：論文集(書籍)内論文  

During mammalian brain development, neural progenitor cells undergo symmetric proliferative divisions followed by asymmetric neurogenic divisions. The division mode of these self-renewing progenitors, together with the cell fate of their progeny, plays critical roles in determining the number of neurons and, ultimately, the size of the adult brain. In the past decade, remarkable progress has been made toward identifying various types of neuronal progenitors. Recent technological advances in live imaging and genetic manipulation have enabled us to link dynamic cell biological events to the molecular mechanisms that control the asymmetric divisions of self-renewing progenitors and have provided a fresh perspective on the modes of division of these progenitors. In addition, comparison of progenitor repertoires between species has provided insight into the expansion and the development of the complexity of the brain during mammalian evolution.

DOI： 10.1101/cshperspect.a015719

Web of Science

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：MARY ANN LIEBERT, INC  

Current stem cell technologies have enabled the induction of cortical progenitors and neurons from embryonic stem cells (ESCs) and induced pluripotent stem cells in vitro. To understand the mechanisms underlying the acquisition of apico-basal polarity and the formation of processes associated with the stemness of cortical cells generated in monolayer culture, here, we developed a novel in utero transplantation system based on the moderate dissociation of adherens junctions in neuroepithelial tissue. This method enables (1) the incorporation of remarkably higher numbers of grafted cells and (2) quantitative morphological analyses at single-cell resolution, including time-lapse recording analyses. We then grafted cortical progenitors induced from mouse ESCs into the developing brain. Importantly, we revealed that the mode of process extension depends on the extrinsic apico-basal polarity of the host epithelial tissue, as well as on the intrinsic differentiation state of the grafted cells. Further, we successfully transplanted cortical progenitors induced from human ESCs, showing that our strategy enables investigation of the neurogenesis of human neural progenitors within the developing mouse cortex. Specifically, human cortical cells exhibit multiple features of radial migration. The robust transplantation method established here could be utilized both to uncover the missing gap between neurogenesis from ESCs and the tissue environment and as an in vivo model of normal and pathological human corticogenesis.

DOI： 10.1089/scd.2013.0251

Web of Science

PubMed

researchmap

記述言語：英語  

A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

DOI： 10.1073/pnas.1216696110

PubMed

researchmap

記述言語：英語  

The mechanisms governing the expansion of neuron number in specific brain regions are still poorly understood. Enlarged neuron numbers in different species are often anticipated by increased numbers of progenitors dividing in the subventricular zone. Here we present live imaging analysis of radial glial cells and their progeny in the ventral telencephalon, the region with the largest subventricular zone in the murine brain during neurogenesis. We observe lineage amplification by a new type of progenitor, including bipolar radial glial cells dividing at subapical positions and generating further proliferating progeny. The frequency of this new type of progenitor is increased not only in larger clones of the mouse lateral ganglionic eminence but also in cerebral cortices of gyrated species, and upon inducing gyrification in the murine cerebral cortex. This implies key roles of this new type of radial glia in ontogeny and phylogeny.

DOI： 10.1038/ncomms3125

Web of Science

PubMed

researchmap

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although the vertebrate brain commonly stems from the neuroepithelial tube, the size and complexity of the pseudostratified organization of the brain have drastically expanded during mammalian evolution, resulting in the formation of a highly folded cortex. Developmental controls of neural progenitor divisions underlie these events. In this review, we introduce recent progress in understanding the control of proliferation and differentiation of neural progenitors from a structural point of view. We particularly shed light on the roles of epithelial structure and mitotic spindle orientation in the generation of various types of neural progenitors.

DOI： 10.1111/j.1440-169X.2012.01345.x

Web of Science

PubMed

researchmap

担当区分：筆頭著者   記述言語：英語  

Radial glia cells function as neural stem cells in the developing brain and generate self-renewing and differentiating daughter cells by asymmetric cell divisions. During these divisions, the apical process or basal process of the elongated epithelial structure is asymmetrically partitioned into daughter cells, depending on developmental contexts. However, in mammalian neurogenesis, the relationship between these subcellular structures and self-renewability is largely unknown. We induced oblique cleavages of radial glia cells to split the apical and basal processes into two daughters, and investigated the fate and morphology of the daughters in slice cultures. We observed that the more basal daughter cell that inherits the basal process self-renews outside of the ventricular zone (VZ), while the more apical daughter cell differentiates. These self-renewing progenitors, termed "outer VZ progenitors," retain the basal but not the apical process, as recently reported for the outer subventricular zone (OSVZ) progenitors in primates (Fietz et al., 2010; Hansen et al., 2010); to self-renew, they require clonal Notch signaling between sibling cells. We also found a small endogenous population of outer VZ progenitors in the mouse embryonic neocortex, consistent with a low frequency of oblique radial glia divisions. Our results describe the general role of the basal process in the self-renewal of neural progenitors and implicate the loss of the apical junctions during oblique divisions as a possible mechanism for generating OSVZ progenitors. We propose that mouse outer VZ progenitors, induced by oblique cleavages, provide a model to study both progenitor self-renewal and OSVZ progenitors.

DOI： 10.1523/JNEUROSCI.4773-10.2011

Web of Science

PubMed

researchmap

記述言語：英語  

During mammalian development, neuroepithelial cells function as mitotic progenitors, which self-renew and generate neurons. Although spindle orientation is important for such polarized cells to undergo symmetric or asymmetric divisions, its role in mammalian neurogenesis remains unclear. Here we show that control of spindle orientation is essential in maintaining the population of neuroepithelial cells, but dispensable for the decision to either proliferate or differentiate. Knocking out LGN, (the G protein regulator), randomized the orientation of normally planar neuroepithelial divisions. The resultant loss of the apical membrane from daughter cells frequently converted them into abnormally localized progenitors without affecting neuronal production rate. Furthermore, overexpression of Inscuteable to induce vertical neuroepithelial divisions shifted the fate of daughter cells. Our results suggest that planar mitosis ensures the self-renewal of neuroepithelial progenitors by one daughter inheriting both apical and basal compartments during neurogenesis.

DOI： 10.1038/ncb1673

Web of Science

PubMed

researchmap

記述言語：英語  

The Start/G1 phase in the cell cycle is an important period during which cells determine their developmental fate, onset of mitotic progression, or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G1 cyclins, has been identified as a positive regulator of cell size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here, we show that Whi3 is phosphorylated by the Ras/cAMP-dependent protein kinase (PKA) and that phosphorylation of Ser-568 in Whi3 by PKA plays an inhibitory role in Whi3 function. Phosphorylation of Whi3 by PKA led to its decreased interaction with CLN3 G1 cyclin mRNA and was required for the promotion of G1/S progression. Furthermore, we demonstrate that the phosphomimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events.

DOI： 10.1074/jbc.M112.402214

Web of Science

PubMed

researchmap

DOI： 10.1073/pnas.1303361110

Web of Science

researchmap

記述言語：英語  

Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G1, but the mechanism by which cells restart from G1 delay remains elusive. We found that Whi3, a negative regulator of G1 cyclin, counteracted Hog1 in the restart from G1 delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G1 delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G1 arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G1 arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism.

DOI： 10.1271/bbb.130260

Web of Science

PubMed

researchmap

記述言語：英語  

In mammalian brain development, neuroepithelial cells act as progenitors that produce self-renewing and differentiating cells. Recent technical advances in live imaging and gene manipulation now enable us to investigate how neural progenitors generate the 2 different types of cells with unprecedented accuracy and resolution, shedding new light on the roles of epithelial structure in cell fate decisions and also on the plasticity of neurogenesis.

DOI： 10.1093/cercor/bhp042

Web of Science

PubMed

researchmap

記述言語：英語  

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general transcriptional corepressor. This repression is mediated by recruitment of the Tup1-Cyc8 complex to target promoters through sequence-specific DNA-binding proteins such as Sko1, which mediates the HOG pathway-dependent regulation. We identified tup1 and cyc8 mutant alleles as the suppressor of osmo-sensitivity of the hog1Delta strain. In these mutants, although the expression of the genes under the control of DNA-binding proteins other than Sko1 was apparently normal, the Sko1-regulated genes GRE2 and AHP1 were derepressed under non-stress conditions, suggesting that the Tup1 and Cyc8 mutant proteins were specifically defective in the repression of the Sko1-dependent genes. Chromatin immunoprecipitation analyses of the GRE2 promoter in the mutants demonstrated that the Sko1-Tup1-Cyc8 complex was localized to the promoter, together with Gcn5/SAGA, suggesting that the erroneous recruitment of SAGA to the promoter led to the derepression.

DOI： 10.1016/j.bbrc.2008.01.033

Web of Science

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The budding yeast Saccharomyces cerevisiae has been used in the fermentation of various kinds of alcoholic beverages. But the effect of ethanol on the cell growth of this yeast is poorly understood. This study shows that the addition of ethanol causes a cell-cycle delay associated with a transient dispersion of F-actin cytoskeleton, resulting in an increase in cell size. We found that the tyrosine kinase Swe1, the negative regulator of Cdc28-Clb kinase, is related to the regulation of cell growth in the presence of ethanol. Indeed, the increase in cell size due to ethanol was partially abolished in the SWE1-deleted cells, and the amount of Swe1 protein increased transiently in the presence of ethanol. These results indicated that Swe1 is involved in cell size control in the presence of ethanol, and that a signal produced by ethanol causes a transient up-regulation of Swe1. Further we investigated comprehensively the ethanol-sensitive strains in the complete set of 4847 non-essential gene deletions and identified at least 256 genes that are important for cell growth in the presence of ethanol.

DOI： 10.1271/bbb.68.968

PubMed

researchmap

担当区分：筆頭著者   記述言語：英語  

Because Ca(2+) signaling of budding yeast, through the activation of calcineurin and the Mpk1/Slt2 mitogen-activated protein kinase cascade, performs redundant function(s) in the events essential for growth, the simultaneous deletion of both these pathways (Delta cnb1 Delta mpk1) leads to lethality. A PTC4 cDNA that encodes a protein phosphatase belonging to the PP2C family was obtained as a high dosage suppressor of the lethality of Delta cnb1 Delta mpk1 strain. Overexpression of PTC4 led to a decrease in the high osmolarity-induced Hog1 phosphorylation, and HOG1 deletion remarkably suppressed the synthetic lethality, indicating an antagonistic role of the high osmolarity glycerol (HOG) pathway and the Ca(2+) signaling pathway in growth regulation. The calcineurin-Crz1 pathway was required for the down-regulation of the HOG pathway. Analysis of the time course of actin polarization, bud formation, and the onset of mitosis in synchronous cell cultures demonstrated that calcineurin negatively regulates actin polarization at the bud site, whereas the HOG pathway positively regulates bud formation at a later step after actin has polarized.

DOI： 10.1074/jbc.M306098200

PubMed

researchmap

記述言語：日本語   出版者・発行元：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/nogeikagaku1924.76.734

CiNii Article

CiNii Books

researchmap

担当区分：筆頭著者   掲載種別：研究論文（学術雑誌）  

researchmap

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：日本生物工学会  

CiNii Article

CiNii Books

researchmap

その他リンク： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=300687

担当区分：筆頭著者   記述言語：英語  

An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects. In a certain genetic background (deltazds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways. Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal. Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition. An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation.

PubMed

researchmap