Outside cells of the preimplantation mouse embryo form the trophectoderm (TE), a process requiring the transcription factor Tead4. Here, we show that transcriptionally active Tead4 can induce Cdx2 and other trophoblast genes in parallel in embryonic stem cells. In embryos, the Tead4 coactivator protein Yap localizes to nuclei of outside cells, and modulation of Tead4 or Yap activity leads to changes in Cdx2 expression. In inside cells, Yap is phosphorylated and cytoplasmic, and this involves the Hippo signaling pathway component Lats. We propose that active Tead4 promotes TE development in outside cells, whereas Tead4 activity is suppressed in inside cells by cell contact- and Lats-mediated inhibition of nuclear Yap localization. Thus, differential signaling between inside and outside cell populations leads to changes in cell fate specification during TE formation.

During pre-implantation mouse development, embryos form blastocysts with establishment of the first two cell lineages: the trophectoderm (TE) which gives rise to the placenta, and the inner cell mass (ICM) which will form the embryo proper. Differentiation of TE is regulated by the transcription factor Caudal-related homeobox 2 (Cdx2), but the mechanisms which act upstream of Cdx2 expression remain unknown. Here we show that the TEA domain family transcription factor, Tead4, is required for TE development. Tead1, Tead2 and Tead4 were expressed in pre-implantation embryos, and at least Tead1 and Tead4 were expressed widely in both TE and ICM lineages. Tead4-/- embryos died at pre-implantation stages without forming the blastocoel. The mutant embryos continued cell proliferation, and adherens junction and cell polarity were not significantly affected. In Tead4-/- embryos, Cdx2 was weakly expressed at the morula stage but was not expressed in later stages. None of the TE specific genes, including Eomes and a Cdx2 independent gene, Fgfr2, was detected in Tead4-/- embryos. Instead, the ICM specific transcription factors, Oct3/4 and Nanog, were expressed in all the blastomeres. Tead4-/- embryos also failed to differentiate trophoblast giant cells when they were cultured in vitro. ES cells with normal in vitro differentiation abilities were established from Tead4-/- embryos. These results suggest that Tead4 has a distinct role from Tead1 and Tead2 in trophectoderm specification of pre-implantation embryos, and that Tead4 is an early transcription factor required for specification and development of the trophectoderm lineage, which includes expression of Cdx2.

Abstract-To investigate the association between cardiovascular mortality and short-term variabilities in blood pressure and heart rate, we performed a long-term prospective study of ambulatory blood pressure monitoring in Ohasama, Japan, starting in 1987. We obtained ambulatory blood pressure and heart rate in 1542 subjects Ն40 years of age. Blood pressure and heart rate variabilities were estimated as a standard deviation measured every 30 minutes by ambulatory monitoring. There were 67 cardiovascular deaths during the follow-up period (meanϭ8.5 years). The Cox proportional hazards model, adjusted for possible confounding factors, demonstrated a significant increase in cardiovascular mortality, with an increase in daytime systolic ambulatory blood pressure variability. A similar trend was observed in daytime diastolic and nighttime ambulatory blood pressures. Cardiovascular mortality rate increased linearly, with a decrease in daytime heart rate variability. Subjects in whom the daytime systolic ambulatory blood pressure variability was larger than third quintile and the daytime heart rate variability was lower than the meanϪSD were at extremely high risk of cardiovascular mortality. The blood pressure and heart rate variabilities obtained every 30 minutes by ambulatory blood pressure monitoring were independent predictors for cardiovascular mortality in the general population.

-/-embryos. Most of the previously identified Yap1-regulated genes were not affected in NIH3T3 cells or mutant mice. In embryos, levels of nuclear Yap1 and Tead1 varied depending on cell type. Strong nuclear accumulation of Yap1 and Tead1 were seen in myocardium, correlating with requirements of Tead1 for proliferation. However, their distribution did not always correlate with proliferation. Taken together, mammalian Tead proteins regulate cell proliferation and contact inhibition as a transcriptional mediator of Hippo signaling, but the mechanisms by which Tead/Yap1 regulate cell proliferation differ depending on the cell type, and Tead, Yap1 and Hippo signaling may play multiple roles in mouse embryos.

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