Biomedical subjects

Kazuki Kurimoto

Publications and source records attributed to Kazuki Kurimoto.

3 recordsLinked to original sources

PubMed2006

Gene expression dynamics during germline specification in mice identified by quantitative single-cell gene expression profiling.

Germ cell fate in mice is induced in proximal epiblast cells at Embryonic Day (E) 6.5 by signaling molecules. Prdm1(also known as Blimp1)-positive lineage-restricted precursors of primordial germ cells (PGCs) initiate the formation of a cluster that differentiates into Dppa3 (also known as stella)-positive PGCs from around E7.0 onwards in the extra-embryonic mesoderm. Around E7.5, these PGCs begin migrating towards the definitive endoderm, with concomitant extensive epigenetic reprogramming. To gain a more precise insight into the mechanism of PGC specification and its subsequent development, we exploited quantitative, single-cell, gene expression profiling to explore gene expression dynamics during the 36 h of PGC differentiation from E6.75 to E8.25, in comparison with the corresponding profiles of somatic neighbors. This analysis revealed that the transitions from Prdm1-positive PGC precursors to Dppa3-positive PGCs and to more advanced migrating PGCs involve a highly dynamic, stage-dependent transcriptional orchestration that begins with the regaining of the pluripotency-associated gene network, followed by stepwise activation of PGC-specific genes, differential repression of the somatic mesodermal program, as well as potential modulations of signal transduction capacities and unique control of epigenetic regulators. The information presented here regarding the cascade of events involved in PGC development should serve as a basis for detailed functional analyses of the gene products associated with this process, as well as for appropriate reconstitution of PGCs and their descendant cells in culture.

Animals↗

PubMed2006

An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis.

A systems-level understanding of a small but essential population of cells in development or adulthood (e.g. somatic stem cells) requires accurate quantitative monitoring of genome-wide gene expression, ideally from single cells. We report here a strategy to globally amplify mRNAs from single cells for highly quantitative high-density oligonucleotide microarray analysis that combines a small number of directional PCR cycles with subsequent linear amplification. Using this strategy, both the representation of gene expression profiles and reproducibility between individual experiments are unambiguously improved from the original method, along with high coverage and accuracy. The immediate application of this method to single cells in the undifferentiated inner cell masses of mouse blastocysts at embryonic day (E) 3.5 revealed the presence of two populations of cells, one with primitive endoderm (PE) expression and the other with pluripotent epiblast-like gene expression. The genes expressed differentially between these two populations were well preserved in morphologically differentiated PE and epiblast in the embryos one day later (E4.5), demonstrating that the method successfully detects subtle but essential differences in gene expression at the single-cell level among seemingly homogeneous cell populations. This study provides a strategy to analyze biophysical events in medicine as well as in neural, stem cell and developmental biology, where small numbers of distinctive or diseased cells play critical roles.

Animals↗

PubMed2005

Crystal structure of the N-terminal RecA-like domain of a DEAD-box RNA helicase, the Dugesia japonica vasa-like gene B protein.

The Dugesia japonica vasa-like gene B (DjVLGB) protein is a DEAD-box RNA helicase of a planarian, which is well known for its strong regenerative capacity. DjVLGB shares sequence similarity to the Drosophila germ-line-specific DEAD-box RNA helicase Vasa, and even higher similarity to its paralogue, mouse PL10. In this study, we solved the crystal structure of the DjVLGB N-terminal RecA-like domain. The overall fold and the structures of the putative ATPase active site of the DjVLGB N-terminal RecA-like domain are similar to those of the previously reported DEAD-box RNA helicase structures. In contrast, the surface structure of the side opposite to the putative ATPase active site is different from those of the other DEAD-box RNA helicases; the characteristic hydrophobic pockets are formed with aromatic and proline residues. These pocket-forming residues are conserved in the PL10-subfamily proteins, but less conserved in the Vasa orthologues and not conserved in the DEAD-box RNA helicases. Therefore, the structural features that we found are characteristic of the PL10-subfamily proteins and might contribute to their biological roles in germ-line development.

Amino Acid Sequence↗