MASAKO TADA
Genome-wide epigenetic reprogramming in the gap period during which cell fate changes drastically during germline development
What is the main role of the germline? The cells that will become germ cells are not predetermined in mammalian embryos. In the early phase of embryonic development, a subset of the pluripotent embryonic cells called epiblasts becomes primordial germ cells (PGCs), which are immature germ cells. The extrinsic key factor inducing PGCs is BMP4. PGCs first appear outside the gonads, and then enter the testis and ovary, where they become sperm and oocytes, respectively. Sperm and oocytes are highly specified cells that are fused through fertilization to become totipotent embryonic cells. After primary cell differentiation into extraembryonic tissues is initiated, the cells that form the embryo are isolated as an inner cell mass (ICM). In implanted blastocyst embryos, these ICM cells differentiate into epiblasts, and PGCs are again developed from epiblasts. The stem cell lineage involved in the cycle of fertilization and germ cell formation is called the germline, and is responsible for continuous maintenance of the life cycle. We are investigating the initialization mechanisms occuring at specific periods in the life cycle of every fetus to transmit only minimized epigenetic information to the next generation.
What is the role of genome-wide epigenetic reprogramming? During germline development, cells pass through multiple differentiation processes. Epigenetic information is reprogrammed during the gap periods prior to the next developmental stage. First, most of the epigenetic information that is specific to the eggs and sperm is reprogrammed as these become totipotent stem cells upon fertilization. In this reprogramming process, which occurs during preimplantation development, maternal and paternal epigenotypes become nearly equivalent, but the genomic imprints are not erased. In addition, the constitutive heterochromatin remains highly inactive. Epiblasts, in contrast, are differentiated from ICM cells and become epigenetically inactivated at high levels. When gastrulation is initiated among a subset of the epiblasts, epiblast cells diverge into mesoderm, endoderm, and PGCs, while the remaining cells become ectoderm. Because the gonads (genital ridge) are primarily formed from mesoderm, PGCs themselves migrate into the genital ridge. Immediately after entering the genital ridge, PGCs are qualitatively changed, as genomic imprints are comprehensively erased and inactive epigenetic marks including DNA cytosine methylation, histone H3 methylation at K9, and X chromosome inactivation are eliminated. Thus there are remarkable differences between the mechanisms for embryonic reprogramming and PGC reprogramming.
Later, PGCs enter meiotic division I and await further epigenetic changes during oogenesis after sexual maturation. On the other hand, sex determination induces Sertoli cell differentiation in the male genital ridge, in which PGCs escape from meiosis but enter mitotic arrest. Therefore, after sex determination, the epigenomes of PGCs begin to be specified depending on the sex of the individual. Once sex is determined, sex-related epigenetic differences are accumulated during oogenesis and spermatogenesis, followed by re-establishment of genomic imprints, most of which are established during oocyte maturation.
What are the major aims of our study? Many phenomena that are fundamental to the mammalian life cycle occur through germline development, such as acquisition of pluripotency, PGC specification, X chromosome inactivation, sex determination, and establishment of genomic imprints. During the developmental gap periods between events, previous epigenotypes are temporarily erased and/or overwritten by multiple events whenever cell statuses are drastically changed from a differentiated state to the next undifferentiated state or from an undifferentiated state to the following differentiated state. Cell fate determining factors for PGCs and ICMs are already identified in mammals. Blimp1 and Prdm14 are crucial transcriptional factors required for epiblasts to become PGCs in mice, while SOX17 performs the function of PRDM14 in humans. Yamanaka’s four factors, Oct4, Sox2, Klf4, and cMyc, are major transcriptional factors involved in maintaining the pluripotency of ICM-derived embryonic stem cells (ESCs). These factors can induce pluripotent stem cells (iPSCs) from somatic tissue cells. However, their downstream factors that add, remove, and edit stage-specific epigenetic profiles have not yet been fully identified.
The major aims of our study are to resolve the following two questions: (1) What is the PGC-specific reprogramming mechanism? (2) What is the mechanism by which the genomes of ESCs and epiblasts can escape fundamental erasure of the key epigenotypes?
How are we addressing this question? To understand how stem cells trigger cell-type specific reprogramming in fetuses and how stem cells maintain cell homeostasis by blocking the next reprogramming wave, we have differentiated mouse embryonic stem cells (ESCs) in vitro in culture medium with various growth factors and/or cell signaling ligands. We are also trying to directly reprogram the epigenomes of ESCs by cell culturing in the medium with small molecules that can positively or negatively alter the functions of chromatin modifying enzymes. Reprogrammed chromosomal regions can be identified as 5-hydroxymethylated regions in germline stem cells.
Mouse ESCs (left) and human iPSCs (right)
H3K9me2/3-positive heterochromain is negative for 5-hydroxymethylation (5hmC) but positive for DNA methylation. Contrary, 5hmC is accumulated at H3K4me2/3-positive euchromatic chromosomal regions in every mouse ESC.
Production of functional human hepatocyte-like cells from stem cells
Why do we need a lot of human adult hepatocytes?
In recent years, her laboratory has also engaged in applied research on stem cells that can support drug discovery and development by producing human adult hepatocyte-like cells from the bipotential hepatic carcinoma cell line HepaRG and pluripotent iPS cells.
