Mammals express four highly conserved TEAD/TEF transcription factors that bind the same DNA sequence, but serve different functions during development. TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with a novel TEAD-binding domain at the amino terminus of YAP65, a powerful transcriptional coactivator. YAP65 interacted specifically with the carboxyl terminus of all four TEAD proteins. Both this interaction and sequence-specific DNA binding by TEAD were required for transcriptional activation in mouse cells. Expression of YAP in lymphocytic cells that normally do not support TEAD-dependent transcription (e.g., MPC11) resulted in up to 300-fold induction of TEAD activity. Conversely, TEAD overexpression squelched YAP activity. Therefore, the carboxy-terminal acidic activation domain in YAP is the transcriptional activation domain for TEAD transcription factors. However, whereas TEAD was concentrated in the nucleus, excess YAP65 accumulated in the cytoplasm as a complex with the cytoplasmic localization protein, 14-3-3. Because TEAD-dependent transcription was limited by YAP65, and YAP65 also binds Src/Yes protein tyrosine kinases, we propose that YAP65 regulates TEAD-dependent transcription in response to mitogenic signals.

Specification of cell lineages in mammals begins shortly after fertilization with formation of a blastocyst consisting of trophectoderm, which contributes exclusively to the placenta, and inner cell mass (ICM), from which the embryo develops. Here we report that ablation of the mouse Tead4 gene results in a preimplantation lethal phenotype, and TEAD4 is one of two highly homologous TEAD transcription factors that are expressed during zygotic gene activation in mouse 2-cell embryos. Tead4 -/-embryos do not express trophectoderm-specific genes, such as Cdx2, but do express ICM-specific genes, such as Oct4 (also known as Pou5f1). Consequently, Tead4-/-morulae do not produce trophoblast stem cells, trophectoderm or blastocoel cavities, and therefore do not implant into the uterine endometrium. However, Tead4 -/-embryos can produce embryonic stem cells, a derivative of ICM, and if the Tead4 allele is not disrupted until after implantation, then Tead4 -/-embryos complete development. Thus, Tead4 is the earliest gene shown to be uniquely required for specification of the trophectoderm lineage.

During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway.

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SUMMARYIt has been suggested that during mouse preimplantation development, the zygotically expressed transcription factor TEAD4 is essential for specification of the trophectoderm lineage required for producing a blastocyst. Here we show that blastocysts can form without TEAD4 but that TEAD4 is required to prevent oxidative stress when blastocoel formation is accompanied by increased oxidative phosphorylation that leads to the production of reactive oxygen species (ROS). Both two-cell and eight-cell Tead4 -/− embryos developed into blastocysts when cultured under conditions that alleviate oxidative stress, and Tead4 −/− blastocysts that formed under these conditions expressed trophectoderm-associated genes. Therefore, TEAD4 is not required for specification of the trophectoderm lineage. Once the trophectoderm was specified, Tead4 was not essential for either proliferation or differentiation of trophoblast cells in culture. However, ablation of Tead4 in trophoblast cells resulted in reduced mitochondrial membrane potential. Moreover, Tead4 suppressed ROS in embryos and embryonic fibroblasts. Finally, ectopically expressed TEAD4 protein could localize to the mitochondria as well as to the nucleus, a property not shared by other members of the TEAD family. These results reveal that TEAD4 plays a crucial role in maintaining energy homeostasis during preimplantation development. KEY WORDS:Tead4, Blastocyst, Trophectoderm, Blastocoel, Oxidative stress, Anti-oxidant, Mouse TEAD4 establishes the energy homeostasis essential for blastocoel formation Kotaro J. Kaneko* and Melvin L. DePamphilis DEVELOPMENT 3681 RESEARCH ARTICLE Tead4 and energy homeostasis that are derived from TE (Tanaka et al., 1998;Yagi et al., 2007). The requirement for TEAD4 during preimplantation development is specific for TE, because Tead4 −/− embryos continue to express genes characteristic of the ICM and can produce embryonic stem cells that are derived from the ICM. Thus, Tead4 is the earliest zygotically expressed transcription factor reported to be essential for blastocoel formation and expression of TE-associated genes. Tead4 might also be required for post-implantation development, because it is expressed selectively in extra-embryonic ectoderm as well as in developing placenta (Jacquemin et al., 1996; Jacquemin et al., 1998).In an effort to understand how TEAD4 specifies TE, we made the surprising discovery that Tead4 −/− embryos could form a blastocoel, express TE-associated genes and produce trophoblast giant cells. Therefore, TEAD4 is not essential for specifying the TE lineage. It is, however, essential for blastocoel formation and expansion, but only under conditions that promote oxidative phosphorylation and therefore oxidative stress. By managing oxidative stress in vitro, the requirement for TEAD4 can be bypassed. In fact, analysis of ectopic Tead4 gene expression as well as ablation of Tead4 gene expression suggested that Tead4 is unique among TEAD members in that TEAD4 can localize to mitochondria and affect mitochondrial activi...

Summary-TEAD2, one of the first transcription factors expressed at the beginning of mammalian development, appears to be required during neural development. For example, Tead2 expression is greatest in the dorsal neural crest where it appears to regulate expression of Pax3, a gene essential for brain development. Consistent with this hypothesis, we found that inactivation of the Tead2 gene in mice significantly increased the risk of exencephaly (a defect in neural tube closure). However, none of the embryos exhibited spina bifida, the major phenotype of Pax3 nullizygous embryos, and expression of Pax3 in E11.5 Tead2 nullizygous embryos was normal. Thus, Tead2 plays a role in neural tube closure that is independent of its putative role in Pax3 regulation. In addition, the risk of exencephaly was greatest with Tead2 nullizygous females, and could be suppressed either by folic acid or pifithrin-α. These results reveal a maternal genetic contribution to neural tube closure, and suggest that Tead2-deficient mice provide a model for anencephaly, a common human birth defect that can be prevented by folic acid. genesis 45:577-587, 2007.

Dietary exposure assessment using food-consumption data and ingredient-use level is essential for assessing the safety of food ingredients. Dietary exposure estimates are compared with safe intake levels, such as the acceptable daily intake (ADI). The ADI is estimated by applying a safety factor to an experimentally determined no-observed-adverse-effect level of a test substance. Two food ingredients classified as emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (P80), received attention recently due to their putative adverse effects on gut microbiota. Because no published dietary exposure estimates for commonly used emulsifiers exist for the US population, the current investigation focused on the estimation of dietary exposure to seven emulsifiers: CMC, P80, lecithin, mono- and diglycerides (MDGs), stearoyl lactylates, sucrose esters, and polyglycerol polyricinoleate. Using maximum-use levels obtained from publicly available sources, dietary exposures to these emulsifiers were estimated for the US population (aged 2 years and older) for two time periods (1999-2002 and 2003-10) using 1- and 2-day food-consumption data from the National Health and Nutrition Examination Survey, and 10-14-day food-consumption data from NPD Group, Inc.'s National Eating Trends - Nutrient Intake Database. Our analyses indicated that among the emulsifiers assessed, lecithin and MDGs have the highest mean exposures at about 60 and about 80 mg kg bw day, respectively, whereas the exposure to CMC is half to one-third that of lecithin or MDGs; and the exposure to P80 is approximately half that of CMC. The review of available safety information such as ADIs established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), in light of our updated dietary exposure estimates for these seven emulsifiers, did not raise safety concerns at the current specified levels of use. Additionally, by examining two time periods (1999-2002, 2003-10), it was concluded that there is no evidence that exposure levels to emulsifiers have substantially increased.

Investigation of the regulatory region of mTEAD-2, a gene expressed at the beginning of mouse pre-implantation development, led to the surprising discovery of another gene only 3.8 kb upstream of mTEAD-2. Here we show that this new gene is a single copy, testis-specific gene called SOGGY: (mSgy) that produces a single, dominant mRNA approximately 1.3 kb in length. It is transcribed in the direction opposite to mTEAD-2, thus placing the regulatory elements of these two genes in close proximity. mSgy contains three methionine codons that could potentially act as translation start sites, but most mSGY protein synthesis in vitro was initiated from the first Met codon to produce a full-length protein, suggesting that mSGY normally consists of 230 amino acids (26.7 kDa). Transcription began at a cluster of nucleotides approximately 150 bp upstream of the first Met codon using a TATA-less promoter contained within the first 0.9 kb upstream. The activity of this promoter was repressed by upstream sequences between -0.9 and -2.5 kb in cells that did not express mSgy, but this repression was relieved in cells that did express mSgy. mSgy mRNA was detected in embryos only after day 15 and in adult tissues only in the developing spermatocytes of seminiferous tubules, suggesting that mSgy is a spermatocyte-specific gene. Since mTEAD-2 and mSgy were not expressed in the same cells, the mSgy/mTEAD-2 locus provides a unique paradigm for differential regulation of gene expression during mammalian development.