Published papers

In this issue of Cell Stem Cell, Pan et al. (2007) and Zhao et al. (2007) report genome-wide histone H3 trimethylations at lysines 4 and 27 in human embryonic stem (ES) cells. Similar gene sets arise via different experimental systems, advancing our understanding of stem cell epigenetics. © 2007 Elsevier Inc. All rights reserved.

Pluripotent stem cell lines with similar phenotypes can be derived from both blastocysts (embryonic stem cells, ESC) and primordial germ cells (embryonic germ cells, EGC). Here, we present a compendium DNA microarray analysis of multiple mouse ESCs and EGCs from different genetic backgrounds (strains 129 and C57BL/6) cultured under standard conditions and in differentiation-promoting conditions by the withdrawal of Leukemia Inhibitory Factor (LIF) or treatment with retinoic acid (RA). All pluripotent cell lines showed similar gene expression patterns, which separated them clearly from other tissue stem cells with lower developmental potency. Differences between pluripotent lines derived from different sources (ESC vs. EGC) were smaller than differences between lines derived from different mouse strains (129 vs. C57BL/6). Even in the differentiation-promoting conditions, these pluripotent cells showed the same general trends of gene expression changes regardless of their origin and genetic background. These data indicate that ESCs and EGCs are indistinguishable based on global gene expression patterns alone. On the other hand, a detailed comparison between a group of ESC lines and a group of EGC lines identified 20 signature genes whose average expression levels were consistently higher in ESC lines, and 84 signature genes whose average expression levels were consistently higher in EGC lines, irrespective of mouse strains. Similar analysis identified 250 signature genes whose average expression levels were consistently higher in a group of 129 cell lines, and 337 signature genes whose average expression levels were consistently higher in a group of C57BL/6 cell lines. Although none of the genes was exclusively expressed in either ESCs versus EGCs or 129 versus C57BL/6, in combination these signature genes provide a reliable separation and identification of each cell type. Differentiation-promoting conditions also revealed some minor differences between the cell lines. For example, in the presence of RA, EGCs showed a lower expression of muscle- and cardiac-related genes and a higher expression of gonad-related genes than ESCs. Taken together, the results provide a rich source of information about the similarities and differences between ESCs and EGCs as well as 129 lines and C57BL/6 lines. Such information will be crucial to our understanding of pluripotent stem cells. The results also underscore the importance of studying multiple cell lines from different strains when making comparisons based on gene expression analysis. © 2007 Elsevier Inc. All rights reserved.

The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named Zscan4a-Zscan4f, three - Zscan4c, Zscan4d, and Zscan4f - encode full-length ORFs with 506 amino acids. Zscan4d is a predominant transcript at the late 2-cell stage, whereas Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development.

The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

To facilitate the analysis of gene regulatory regions of the mouse genome, we developed a CisView (http:x//lgsun.grc.nia.nih.gov/cisview), a browser and database of genome-wide potential transcription factor binding sites (TFBSs) that were identified using 134 position-weight matrices and 219 sequence patterns from various sources and were presented with the information about sequence conservation, neighboring genes and their structures, GO annotations, protein domains, DNA repeats and CpG islands. Analysis of the distribution of TFBSs revealed that many TFBSs (N = 145) were over-represented near transcription start sites. We also identified potential cis-regulatory modules (CRMs) defined as clusters of conserved TFBSs in the entire mouse genome. Out of 739 074 CRMs, 157 442 had a significantly higher regulatory potential score than semi-random sequences generated with a 3rd-order Markov process. The CisView browser provides a user-friendly computer environment for studying transcription regulation on a whole-genome scale and can also be used for interpreting microarray experiments and identifying putative targets of transcription factors. © The Author 2006. Kazusa DNA Research Institute.

POU transcription factor Pou5f1 (Oct3/4) is required to maintain ES cells in an undifferentiated state. Here we show that global expression profiling of Oct3/4-manipulated ES cells delineates the downstream target genes of Oct3/4. Combined with data from genome-wide chromatin-immunoprecipitation (ChIP) assays, this analysis identifies not only primary downstream targets of Oct3/4, but also secondary or tertiary targets. Furthermore, the analysis also reveals that downstream target genes are regulated either positively or negatively by Oct3/4. Identification of a group of genes that show both activation and repression depending on Oct3/4 expression levels provides a possible mechanism for the requirement of appropriate Oct3/4 expression to maintain undifferentiated ES cells. As a proof-of-principle study, one of the downstream genes, Tcl1, has been analyzed in detail. We show that Oct3/4 binds to the promoter region of Tcl1 and activates its transcription. We also show that Tcl1 is involved in the regulation of proliferation, but not differentiation, in ES cells. These findings suggest that the global expression profiling of gene-manipulated ES cells can help to delineate the structure and dynamics of gene regulatory networks.

Large-scale systematic gene expression analyses of early embryos and stem cells provide useful information to identify genes expressed differentially or uniquely in these cells. We review the current status of various approaches applied to preimplantation embryos and stem cells: expressed sequence tag, serial analysis of gene expression, differential display, massively parallel signature sequencing, DNA microarray (DNA chip) analysis, and chromatin-immunoprecipitation microarrays. We also discuss the biological questions that can only be addressed by the analysis of global gene expression patterns, such as so-called stemness and developmental potency. As the emphasis now shifts from expression profiling to functional studies, we review the genome-scale functional studies of genes: expression cloning, gene trapping, RNA interference, and gene disruptions. Finally, we discuss the future clinical application of such methodologies.

Although the POU transcription factor Oct3/4 is pivotal in maintaining self renewal of embryonic stem (ES) cells, little is known of its molecular mechanisms. We previously reported that the N-terminal transactivation domain of Oct3/4 is required for activation of Lefty1 expression (H. Niwa, S. Masui, I. Chambers, A. G. Smith, and J. Miyazaki, Mol. Cell. Biol. 22:1526-1536, 2002). Here we test whether Lefty1 is a direct target of Oct3/4. We identified an ES cell-specific enhancer upstream of the Lefty1 promoter that contains binding sites for Oct3/4 and Sox2. Unlike other known Oct3/4-Sox2-dependent enhancers, however, this enhancer element could not be activated by Oct3/4 and Sox2 in differentiated cells. By functional screening of ES-specific transcription factors, we found that Krüppel-like factor 4 (Klf4) cooperates with Oct3/4 and Sox2 to activate Lefty1 expression, and that Klf4 acts as a mediating factor that specifically binds to the proximal element of the Lefty1 promoter. DNA microarray analysis revealed that a subset of putative Oct3/4 target genes may be regulated in the same manner. Our findings shed light on a novel function of Oct3/4 in ES cells. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Analysis of gene expression changes during preimplantation development by quantitative reverse transcription-polymerase chain reaction (Q-PCR) requires appropriate internal standards. Ideally, such a gene should show a constant level of transcripts per embryo across all preimplantation stages from unfertilized eggs to blastocysts. By analysing the microarray-based gene expression profiles of preimplantation embryos, it was found that a conserved helix-loop-helix ubiquitous kinase gene (Chuk, also known as IκB kinase α, IKKα or IKK1) satisfied this criterion. To test the utility of this gene as an internal standard for Q-PCR, the expression levels of two known genes (Nalp5/Mater, Pou5f1/Oct3/Oct4) were normalized by Chuk and other housekeeping genes (Actb, Gapdh, Eef1a1, and H2afz) and demonstrated that the former was more consistent with the expression patterns obtained by a whole-mount in-situ hybridization than those reported previously with the latter. It is concluded that Chuk, unlike other commonly used normalization controls, is a reliable and suitable internal standard for measuring gene expression levels by Q-PCR in mouse oocytes and preimplantation embryos.

The laboratory mouse model plays important roles in our understanding of early mammalian development and provides an invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. A comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over the past two decades, reveal even further the value of the mouse models. Here, the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells is reviewed and future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as a snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, including a bird's-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks.

Preimplantation development is marked by four major events: the transition of maternal transcripts to zygotic transcripts, compaction, the first lineage differentiation into inner cell mass and trophectoderm, and implantation. The scarcity of the materials of preimplantation embryos, both in size (diameter < 100 microm) and in quantity (only a few to tens of oocytes from each ovulation), has hampered molecular analysis of preimplantation embryos. Recent progress in RNA amplification methods and microarray platforms, including genes unique to preimplantation embryos, allow us to apply global gene expression profiling to the study of preimplantation embryos. Our gene expression profiling during preimplantation development revealed the distinctive patterns of maternal RNA degradation and embryonic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA). The second wave, mid-preimplantation gene activation (MGA), contributes dramatic morphological changes during late preimplantation development. Further expression profiling of embryos treated with inhibitors of transcription or translation revealed that the translation of maternal RNA is required for the initiation of ZGA, suggesting a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. To date, several reports of microarray experiments using mouse and human preimplantation embryos have been published. The identification of a large number of genes and multiple signaling pathways involved at each developmental stage by such global gene expression profiling accelerates understanding of molecular mechanisms underlining totipotency/pluripotency and programs of early mammalian development.

In our earlier attempt to identify genes involved in the maintenance of cellular pluripotency, we found that KH-domain protein Embryonal stem cell-specific gene 1 (Esg1) showed similar expression patterns to those of Oct3/4 (Pou5f1), whereas the forced repression of Oct3/4 in mouse embryonic stem cells immediately downregulated the expression of Esg1. Here we further confirm this overlap by in situ hybridization and immunohistochemical analyses. Both Esg1 transcript and protein exist in the egg and preimplantation embryos. At embryonic day 3.5, blastocyst stage, however, ESG1 protein was more abundant in the inner cell mass (ICM) than in trophectoderm (TE), whereas Esg1 transcript was detected in both the ICM and the TE, particularly in the polar trophectoderm. The presence of an RNA-binding KH-domain in ESG1 led us to search for and identify 902 target transcripts by microarray analysis of immunoprecipitated ESG1 complex. Interaction of 20 target mRNA with ESG1, including Cdc25a, Cdc42, Ezh2, Nfyc and Nr5a2. was further validated by reverse transcriptase-polymerase chain reaction of the immunoprecipitation material, supporting the notion that ESG1 is an RNA-binding protein which associates with specific target transcripts. © 2006 Japanese Society of Developmental Biologists.

A series of Ca2+ oscillations during mammalian fertilization is necessary and sufficient to stimulate meiotic resumption and pronuclear formation. It is not known how effectively development continues in the absence of the initial Ca2+ signal. We have triggered parthenogenetic egg activation with cycloheximide that causes no Ca2+ increase, with ethanol that causes a single large Ca2+ increase, or with Sr2+ that causes Ca2+ oscillations. Eggs were co-treated with cytochalasin D to make them diploid and they formed pronuclei and two-cell embryos at high rates with each activation treatment. However, far fewer of the embryos that were activated by cycloheximide reached the blastocyst stage compared to those activated by Sr2+ or ethanol. Any cycloheximide-activated embryos that reached the blastocyst stage had a smaller inner cell mass number and a greater rate of apoptosis than Sr2+ -activated embryos. The poor development of cycloheximide-activated embryos was due to the lack of Ca2+ increase because they developed to blastocyst stages at high rates when co-treated with Sr2+ or ethanol. Embryos activated by either Sr2+ or cycloheximide showed similar signs of initial embryonic genome activation (EGA) when measured using a reporter gene. However, microarray analysis of gene expression at the eight-cell stage showed that activation by Sr2+ leads to a distinct pattern of gene expression from that seen with embryos activated by cycloheximide. These data suggest that activation of mouse eggs in the absence of a Ca2+ signal does not affect initial parthenogenetic events, but can influence later gene expression and development. © 2006 Society for Reproduction and Fertility.

To understand global features of gene expression changes during in vitro neural differentiation, we carried out the microarray analysis of embryonic stem cells (ESCs), embryonal carcinoma cells, and adult neural stem/progenitor (NS) cells. Expression profiling of ESCs during differentiation in monolayer culture revealed three distinct phases: undifferentiated ESCs, primitive ectoderm-like cells, and neural progenitor cells. Principal component (PC) analysis revealed that these cells were aligned on PC1 over the course of 6 days. This PC1 represents approximately 4,000 genes, the expression of which increased with neural commitment/differentiation. Furthermore, NS cells derived from adult brain and their differentiated cells were positioned along this PC axis further away from undifferentiated ESCs than embryonic stem-derived neural progenitors. We suggest that this PC1 defines a path to neural fate, providing a scale for the degree of commitment/differentiation. ©AlphaMed Press.

Thyroid hormone (TH) controlled gene expression profiles have been studied in the tail, hind limb and brain tissues during TH-induced and spontaneous Xenopus laevis metamorphosis. Amplified cRNA probes mixed with a universal standard were hybridized to a set of 21,807-sense strand 60-mer oligonucleotides on each slide representing the entries in X. laevis UniGene Build 48. Most of the up-regulated genes in hind limb and brain are the same. This reflects in part the fact that the initial response to TH induction in both tissues is cell proliferation. A large number of up-regulated genes in the limb and brain programs encode common components of the cell cycle, DNA and RNA metabolism, transcription and translation. Notch is one of the few genes that is differentially expressed exclusively in the brain in the first 48 h of TH induction studied in these experiments. The TH-induced gene expression changes in the tail are different from the limb and brain programs. Distinct muscle and fibroblast programs were identified in the tail. Dying muscle fibers in tail (marked by active caspase-3) up-regulate a group of genes that include proteolytic enzymes. At the climax of metamorphosis, tail muscle down-regulates more than half of the genes that encode the glycolytic enzymes in the cytoplasm and the tricarboxylic acid pathway and all five complexes of the electron transport system in mitochondria. These changes in gene expression precede the activation of caspase-3. Some of these same energy metabolism-related genes are up-regulated in the limb and brain programs by TH. A prominent feature of the tail fibroblasts is the down-regulation of several collagen and other extra cellular matrix genes and the up-regulation of hydrolytic enzymes that are responsible for dissolving the notochord and resorbing the tail. © 2005 Elsevier Inc. All rights reserved.

The International Symposium entitled "Germ Cells, Epigenetics, Reprogramming, and Embryonic Stem Cells" was organized by Norio Nakatsuji (Kyoto University) and Hiromitsu Nakauchi (University of Tokyo) in Kyoto, Japan (November 15-18, 2005). The meeting provided an overview of this important research area and highlighted recent advances. © 2006 Elsevier Inc.

The La protein is a target of autoantibodies in patients suffering from Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La +/- intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM.

Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos (∼60 μm diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the inner cell mass (ICM) and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos.

Networks of transcription factors (TFs) are thought to determine and maintain the identity of cells. Here we systematically repressed each of 100 TFs with shRNA and carried out global gene expression profiling in mouse embryonic stem (ES) cells. Unexpectedly, only the repression of a handful of TFs significantly affected transcriptomes, which changed in two directions/ trajectories: one trajectory by the repression of either Pou5f1 or Sox2; the other trajectory by the repression of either Esrrb, Sall4, Nanog, or Tcfap4. The data suggest that the trajectories of gene expression change are already preconfigured by the gene regulatory network and roughly correspond to extraembryonic and embryonic fates of cell differentiation, respectively. These data also indicate the robustness of the pluripotency gene network, as the transient repression of most TFs did not alter the transcriptomes.