Aim of our team is to develop technologies and experimental tools/ resources for characterization of 'genotype, phenotype and epigenotype' of biological resources. Through these efforts, we would extend utilities of bioresources collected at RIKEN BRC. In order to achieve this goal, our team uses various approaches including genetics, genomics and transgenic/gene targeting technologies. Based on these technologies and resources development, we will establish analytical platform for analyses of dynamic nature of mammalian genome in response to changes in various environmental factors. We focus on early mouse developmental processes, in particular, development and genomic reprogramming process of pluripotent embryonic cells and primordial germ cells. Also, we will explore how environmental factors influence on development and growth of organisms, or on the onset of disease condition.
･ Visualization, purification and micro-scale analyses of specific cell lineage
（Lineage-specific reporter mouse strains，minuscule-scale transcriptome and epigenome analysis）
･ Analytical tools for epigenetic regulatory mechanisms operating during genomic reprogramming process ( new stem cell lines, novel techniques for DNA methylation analysis, Methylation-specific FISH )
･ Development of resources and technologies for the analysis of wild-derived inbred mouse strains ( BAC libraries, BAC transgenic mice, SNP analytical techniques )
･ Analysis of functional processes in live animals by fluorescent in vivo imaging ( Fluorescent reporter transgenic mice, intravital microscope )
1. Studies of early mammalian development through the analysis of t-complex mutants.
2. Cell lineage-specific analysis of gene expression in pluripotent embryonic cells and germ cells.
3. Studies on dynamics of epigenetic changes during mammalian development.
4. Functional genomic analysis of wild-derived inbred mouse strains collected at BRC using novel research resources.
5. Phenotyping mouse mutants with in vivo imaging technology.
1. Study on developmental mutant showing defects in proliferation and differentiation of pluripotent embryonic cells.
tw5 mutant embryo at gastrulation stage（a; normal、b; mutant）
tclw5 is a t-complex recessive lethal mutation of the tw5-haplotype. Since tw5/tw5 embryos die soon after implantation, the tclw5 gene is thought to play an important role in early embryogenesis. tw5 homozygotes do not survive past the gastrulation stage due to extensive death of the embryonic ectoderm, a pluripotent cell type giving rise to all three germ layers at subsequent developmental stages. Using this mutant strain, molecular mechanism governing development of this pluripotent cell type will be sought.
2. Visualization of pluripotent embryonic stem cells, germ cells,
and genome wide analysis of gene expression in these cells.
Comprehensive analysis of cell lineage-specific gene expression
In developing mammalian embryos, there exist pluripotent stem cells, giving rise to all somatic cells as well as germ line cells. Primordial germ cell (PGC) is the cell-type appeared first in the germ cell lineage, sharing many features with the embryonic stem cells. We have established systematic methodologies to analyze PGCs and related embryonic cells: PGCs were purified from transgenic mouse embryos, in which the PGCs were marked by GFP-reporter expression. Transcriptomes of these cells were explored by EST analyses from PGC cDNA libraries and microarray.
3. Visualization of DNA methylation patterns in living ES cells; Dynamics of nuclear architecture reorganization during ES cell differentiation
Nuclear architecture is dynamically changed during cell differentiation
Changes in nuclear organization and the epigenetic state of the genome are important driving forces for developmental gene expression. However, a strategy that allows simultaneous visualization of the dynamics of the epigenomic state and nuclear structure has been lacking to date. The methylated DNA binding domain (MBD) and the nuclear localization signal (nls) sequence coding for human methyl CpG-binding domain protein 1 (MBD1) were fused to the enhanced green fluorescent protein (EGFP) reporter gene.The EGFP-MBD-nls protein was used to follow DNA methylation in situ under physiological conditions. We can also monitor the formation and rearrangement of methylated heterochromatin using EGFP-MBD-nls during cell differentiation.