Konstantinos Anastassiadis

Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4

Oct4 is a mammalian POU transcription factor expressed by early embryo cells and germ cells. We report that the activity of Oct4 is essential for the identity of the pluripotential founder cell population in the mammalian embryo. Oct4-deficient embryos develop to the blastocyst stage, but the inner cell mass cells are not pluripotent. Instead, they are restricted to differentiation along the extraembryonic trophoblast lineage. Furthermore, in the absence of a true inner cell mass, trophoblast proliferation is not maintained in Oct4-/- embryos. Expansion of trophoblast precursors is restored, however, by an Oct4 target gene product, fibroblast growth factor-4. Therefore, Oct4 also determines paracrine growth factor signaling from stem cells to the trophectoderm.

Expression Pattern of Oct-4 in Preimplantation Embryos of Different Species

Abstract POU transcription factors are involved in transcriptional regulation during early embryonic development and cell differentiation. Oct-4, a member of this family, has been shown to be under strict regulation during murine development. The expression of Oct-4 correlates with the undifferentiated cell phenotype of the mouse preimplantation embryo. In this study, expression of a gene construct consisting of selected parts of the region upstream from the murine Oct-4 gene as promoter/enhancer, enhanced green fluorescent protein (EGFP) as reporter and the five exons of the murine Oct-4 gene (GOF18-ΔPE EGFP) was evaluated in murine, porcine, and bovine preimplantation embryos. For comparison, expression of the endogenous Oct-4 gene was also analyzed in all three species by immunocytochemistry. The transgene construct was microinjected into zygotes cultured in vitro to various developmental stages. The EGFP fluorescence was visualized in developing embryos by excitation with blue light at different days following microinjection and showed similar expression patterns in all three species. Most embryos displayed a mosaic pattern of transgene expression. The EGFP fluorescence was not restricted to the inner cell mass (ICM) but was also seen in trophoblastic cells. An affinity-purified polyclonal antibody specific to Oct-4 was used for immunocytochemical analysis of in vivo- and in vitro-derived bovine and porcine blastocysts and also of in vivo-derived murine blastocysts. In the in vivo-derived murine embryos, Oct-4 protein was detectable in the ICM but not the trophectoderm, whereas in porcine and bovine blastocysts, derived in vivo or in vitro, Oct-4 protein was detected in both the ICM and the trophectoderm. Thus, in the two large animal species, Oct-4 expression from the endogenous gene was clearly not restricted to the pluripotent cells of the early embryo. These results show that Oct-4 regulation differs between these species and that the presence of Oct-4 protein may not be sufficient for selection of undifferentiated cell lines in domestic animals.

A mouse model for hereditary thyroid dysgenesis and cleft palate.

Alteration of thyroid gland morphogenesis (thyroid dysgenesis) is a frequent human malformation. Among the one in three to four thousand newborns in which congenital hypothyroidism is detected, 80% have either an ectopic, small and sublingual thyroid, or have no thyroid tissue. Most of these cases appear sporadically, although a few cases of recurring familial thyroid dysgenesis have been described. The lack of evidence for hereditary thyroid dysgenesis may be due to the severity of the hypothyroid phenotype. Neonatal screening and early thyroid hormone therapy have eliminated most of the clinical consequences of hypothyroidism such that the heritability of this condition may become apparent in the near future. We have recently cloned cDNA encoding a forkhead domain-containing transcription factor, TTF-2, and have located the position of the gene, designated Titf2, to mouse chromosome 4 (ref. 3). Titf2 is expressed in the developing thyroid, in most of the foregut endoderm and in craniopharyngeal ectoderm, including Rathkes pouch. Expression of Titf2 in thyroid cell precursors is down-regulated as they cease migration, suggesting that this factor is involved in the process of thyroid gland morphogenesis. Here we show that Titf2-null mutant mice exhibit cleft palate and either a sublingual or completely absent thyroid gland. Thus, mutation of Titf2 –/– results in neonatal hypothyroidism that shows similarity to thyroid dysgenesis in humans.

A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.

Pluripotent embryonic stem cells (ESCs) maintain self-renewal while ensuring a rapid response to differentiation cues. The identification of genes maintaining ESC identity is important to develop these cells for their potential therapeutic use. Here we report a genome-scale RNAi screen for a global survey of genes affecting ESC identity via alteration of Oct4 expression. Factors with the strongest effect on Oct4 expression included components of the Paf1 complex, a protein complex associated with RNA polymerase II. Using a combination of proteomics, expression profiling, and chromatin immunoprecipitation, we demonstrate that the Paf1C binds to promoters of key pluripotency genes, where it is required to maintain a transcriptionally active chromatin structure. The Paf1C is developmentally regulated and blocks ESC differentiation upon overexpression, and the knockdown in ESCs causes expression changes similar to Oct4 or Nanog depletions. We propose that the Paf1C plays an important role in maintaining ESC identity.

Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development

Epigenesis is the process whereby the daughters of a dividing cell retain a chromatin state determined before cell division. The best-studied cases involve the inheritance of heterochromatic chromosomal domains, and little is known about specific gene regulation by epigenetic mechanisms. Recent evidence shows that epigenesis pivots on methylation of nucleosomes at histone 3 lysines 4, 9 or 27. Bioinformatics indicates that mammals have several enzymes for each of these methylations, including at least six histone 3 lysine 4 methyltransferases. To look for evidence of gene-specific epigenetic regulation in mammalian development, we examined one of these six, Mll2, using a multipurpose allele in the mouse to ascertain the loss-of-function phenotype. Loss of Mll2 slowed growth, increased apoptosis and retarded development, leading to embryonic failure before E11.5. Using chimera experiments, we demonstrated that Mll2 is cell-autonomously required. Evidence for gene-specific regulation was also observed. Although Mox1 and Hoxb1 expression patterns were correctly established, they were not maintained in the absence of Mll2, whereas Wnt1 and Otx2 were. The Mll2 loss-of-function phenotype is different from that of its sister gene Mll, and they regulate different Hox complex genes during ES cell differentiation. Therefore, these two closely related epigenetic factors play different roles in development and maintain distinct gene expression patterns. This suggests that other epigenetic factors also regulate particular patterns and that development entails networks of epigenetic specificities.

A Localized Wnt Signal Orients Asymmetric Stem Cell Division in Vitro

Stem cells orient cell division and generate a distinct cell fate based on position relative to the Wnt source. Developmental signals such as Wnts are often presented to cells in an oriented manner. To examine the consequences of local Wnt signaling, we immobilized Wnt proteins on beads and introduced them to embryonic stem cells in culture. At the single-cell level, the Wnt-bead induced asymmetric distribution of Wnt–β-catenin signaling components, oriented the plane of mitotic division, and directed asymmetric inheritance of centrosomes. Before cytokinesis was completed, the Wnt-proximal daughter cell expressed high levels of nuclear β-catenin and pluripotency genes, whereas the distal daughter cell acquired hallmarks of differentiation. We suggest that a spatially restricted Wnt signal induces an oriented cell division that generates distinct cell fates at predictable positions relative to the Wnt source.

Current issues in mouse genome engineering

The mouse is the foremost vertebrate experimental model because its genome can be precisely and variously engineered. Now that the mouse genome has been sequenced and annotated, the task of mutating each gene is feasible, and an international cooperation is providing mutated embryonic stem cells and mice as readily available resources. Because these resources will change biomedical research, decisions about their nature will have far-reaching effects. It is therefore timely to consider topical issues for mouse genome engineering, such as the background genotype; homologous, site-specific and transpositional recombination; conditional mutagenesis; RNA-mediated interference; and functional genomics with embryonic stem cells.

Targeted Mutation Reveals Essential Functions of the Homeodomain Transcription Factor Shox2 in Sinoatrial and Pacemaking Development

Background— Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. Methods and Results— To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2−/− embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. Conclusions— From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

MLL2 Is Required in Oocytes for Bulk Histone 3 Lysine 4 Trimethylation and Transcriptional Silencing

Conditional knockout mouse strategies identify the histone methyltranferase MLL2 as a key player in epigenetic reprogramming of female gametes.

Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant

Trimethylation of histone H3 lysine 4 (H3K4me3) at the promoters of actively transcribed genes is a universal epigenetic mark and a key product of Trithorax group action. Here, we show that Mll2, one of the six Set1/Trithorax-type H3K4 methyltransferases in mammals, is required for trimethylation of bivalent promoters in mouse embryonic stem cells. Mll2 is bound to bivalent promoters but also to most active promoters, which do not require Mll2 for H3K4me3 or mRNA expression. By contrast, the Set1 complex (Set1C) subunit Cxxc1 is primarily bound to active but not bivalent promoters. This indicates that bivalent promoters rely on Mll2 for H3K4me3 whereas active promoters have more than one bound H3K4 methyltransferase, including Set1C. Removal of Mll1, sister to Mll2, had almost no effect on any promoter unless Mll2 was also removed, indicating functional backup between these enzymes. Except for a subset, loss of H3K4me3 on bivalent promoters did not prevent responsiveness to retinoic acid, thereby arguing against a priming model for bivalency. In contrast, we propose that Mll2 is the pioneer trimethyltransferase for promoter definition in the naïve epigenome and that Polycomb group action on bivalent promoters blocks the premature establishment of active, Set1C-bound, promoters.