DNA甲基化与脊椎动物胚胎发育

DNA甲基化是指DNA甲基转移酶(DNMT)将DNA序列中的5′胞嘧啶转变为5′甲基胞嘧啶的化学修饰, 可以调控基因的时空特异性表达, 从而影响细胞命运决定和分化等生物学过程。近年来研究发现, DNA甲基化在脊椎动物胚胎早期发育中有重要作用, Dnmt基因的缺失会影响胚胎早期发育和多个器官的形成及分化, 如胚胎早期致死、内脏器官和神经系统终末分化缺陷以及血液发生紊乱等。文章总结了DNA甲基化转移酶在小鼠和斑马鱼发育过程中的动态变化, 并系统阐述了DNA甲基化在胚胎早期发育和器官发生中的作用, 重点揭示DNA 甲基化转移酶与组蛋白甲基化转移酶如何协同调控DNA甲基化从而影响基因转录的分子机理。DNA甲基化作为一种关键的表观遗传学因素, 全面系统地理解其在胚胎发育过程中的作用机制对靶向治疗人类相关疾病有一定的理论指导意义。     

Genetic mosaic analysis reveals that GATA-4 is required for proper differentiation of mouse gastric epithelium.

During mouse embryogenesis GATA-4 is expressed first in primitive endoderm and then in definitive endoderm derivatives, including glandular stomach and intestine. To explore the role of GATA-4 in specification of definitive gastric endoderm, we generated chimeric mice by introducing Gata4(-/-) ES cells into ROSA26 morulae or blastocysts. In E14.5 chimeras, Gata4(-/-) cells were represented in endoderm lining the proximal and distal stomach. These cells expressed early cytodifferentiation markers, including GATA-6 and ApoJ. However, by E18.5, only rare patches of Gata4(-/-) epithelium were evident in the distal stomach. This heterotypic epithelium had a squamous morphology and did not express markers associated with differentiation of gastric epithelial cell lineages. Sonic Hedgehog, an endoderm-derived signaling molecule normally down-regulated in the distal stomach, was overexpressed in Gata4(-/-) cells. We conclude that GATA-4-deficient cells have an intrinsic defect in their ability to differentiate. Similarities in the phenotypes of Gata4(-/-) chimeras and mice with other genetically engineered mutations that affect gut development suggest that GATA-4 may be involved in the gastric epithelial response to members of the TGF-beta superfamily.     

Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells

It has been reported that DNA methyltransferase 1-deficient (Dnmt1-/-) embryonic stem (ES) cells are hypomethylated (20% CpG methylation) and die through apoptosis when induced to differentiate. Here, we show that Dnmt[3a-/-,3b-/-] ES cells with just 0.6% of their CpG dinucleotides behave differently: the majority of cells within the culture are partially or completely blocked in their ability to initiate differentiation, remaining viable while retaining the stem cell characteristics of alkaline phosphatase and Oct4 expression. Restoration of DNA methylation levels rescues these defects. Severely hypomethylated Dnmt[3a-/-,3b-/-] ES cells have increased histone acetylation levels, and those cells that can differentiate aberrantly express extraembryonic markers of differentiation. Dnmt[3a-/-,3b-/-] ES cells with >10% CpG methylation are able to terminally differentiate, whereas Dnmt1-/- ES cells with 20% of the CpG methylated cannot differentiate. This demonstrates that successful terminal differentiation is not dependent simply on adequate methylation levels. There is an absolute requirement that the methylation be delivered by the maintenance enzyme Dnmt1.     

BMP4 induces primitive endoderm but not trophectoderm in monkey embryonic stem cells

Monkey embryonic stem (ES) cells share similar characteristics to human ES cells and provide a primate model of allotransplantation, which allows to validate efficacy and safety of cell transplantation therapy in regenerative medicine. Bone morphogenetic protein 4 (BMP4) is known to promote trophoblast differentiation in human ES cells in contrast to mouse ES cells where BMP4 synergistically maintains self-renewal with leukemia inhibitory factor (LIF), which represents a significant difference in signal transduction of self-renewal and differentiation between murine and human ES cells. As the similarity of the differentiation mechanism between monkey and human ES cells is of critical importance for their use as a primate model system, we investigated whether BMP4 induces trophoblast differentiation in monkey ES cells. Interestingly, BMP4 did not induce trophoblast differentiation, but instead induced primitive endoderm differentiation. Prominent downregulation of Sox2, which plays a pivotal role not only in pluripotency but also placenta development, was observed in cells treated with BMP4. In addition, upregulation of Hand1, Cdx2, and chorionic gonadotropin beta (CG-beta), which are markers of trophoblast, was not observed. In contrast, BMP4 induced significant upregulation of Gata6, Gata4, and LamininB1, suggesting differentiation into the primitive endoderm, visceral endoderm, and parietal endoderm, respectively. The threshold of BMP4 activity was estimated as about 10 ng/mL. These findings suggest that BMP4 induced differentiation into the primitive endoderm lineage but not into trophoblast in monkey ES cells.     

A system to enrich for primitive streak-derivatives, definitive endoderm and mesoderm, from pluripotent cells in culture

Two lineages of endoderm develop during mammalian embryogenesis, the primitive endoderm in the pre-implantation blastocyst and the definitive endoderm at gastrulation. This complexity of endoderm cell populations is mirrored during pluripotent cell differentiation in vitro and has hindered the identification and purification of the definitive endoderm for use as a substrate for further differentiation. The aggregation and differentiation of early primitive ectoderm-like (EPL) cells, resulting in the formation of EPL-cell derived embryoid bodies (EPLEBs), is a model of gastrulation that progresses through the sequential formation of primitive streak-like intermediates to nascent mesoderm and more differentiated mesoderm populations. EPL cell-derived EBs have been further analysed for the formation of definitive endoderm by detailed morphological studies, gene expression and a protein uptake assay. In comparison to embryoid bodies derived from ES cells, which form primitive and definitive endoderm, the endoderm compartment of embryoid bodies formed from EPL cells was comprised almost exclusively of definitive endoderm. Definitive endoderm was defined as a population of squamous cells that expressed Sox17, CXCR4 and Trh, which formed without the prior formation of primitive endoderm and was unable to endocytose horseradish peroxidase from the medium. Definitive endoderm formed in EPLEBs provides a substrate for further differentiation into specific endoderm lineages; these lineages can be used as research tools for understanding the mechanisms controlling lineage establishment and the nature of the transient intermediates formed. The similarity between mouse EPL cells and human ES cells suggests EPLEBs can be used as a model system for the development of technologies to enrich for the formation of human ES cell-derived definitive endoderm in the future.     

Establishment and maintenance of genomic methylation patterns in mouse embryonic stem cells by Dnmt3a and Dnmt3b

We have previously shown that the DNA methyltransferases Dnmt3a and Dnmt3b carry out de novo methylation of the mouse genome during early postimplantation development and of maternally imprinted genes in the oocyte. In the present study, we demonstrate that Dnmt3a and Dnmt3b are also essential for the stable inheritance, or “maintenance,” of DNA methylation patterns. Inactivation of both Dnmt3a and Dnmt3b in embryonic stem (ES) cells results in progressive loss of methylation in various repeats and single-copy genes. Interestingly, introduction of the Dnmt3a, Dnmt3a2, and Dnmt3b1 isoforms back into highly demethylated mutant ES cells restores genomic methylation patterns; these isoforms appear to have both common and distinct DNA targets, but they all fail to restore the maternal methylation imprints. In contrast, overexpression of Dnmt1 and Dnmt3b3 failed to restore DNA methylation patterns due to their inability to catalyze de novo methylation in vivo. We also show that hypermethylation of genomic DNA by Dnmt3a and Dnmt3b is necessary for ES cells to form teratomas in nude mice. These results indicate that genomic methylation patterns are determined partly through differential expression of different Dnmt3a and Dnmt3b isoforms.     

Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors acquire the character of XEN cells

Background  

Three types of cell lines have been established from mouse blastocysts: embryonic stem (ES) cells, trophoblast stem (TS) cells, and extra-embryonic endoderm (XEN) cells, which have the potential to differentiate into their respective cognate lineages. ES cells can differentiate in vitro not only into somatic cell lineages but into extra-embryonic lineages, including trophectoderm and extra-embryonic endoderm (ExEn) as well. TS cells can be established from ES cells by the artificial repression of Oct3/4 or the upregulation of Cdx2 in the presence of FGF4 on feeder cells. The relationship between these embryo-derived XEN cells and ES cell-derived ExEn cell lines remains unclear, although we have previously reported that overexpression of Gata4 or Gata6 induces differentiation of mouse ES cells into extra-embryonic endoderm in vitro.     

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.