The earliest cell differentiation event in mammalian development occurs during preimplantation development when the outer blastomeres of the embryo form a monolayer of epithelial cells

INTRODUCTION The earliest cell differentiation event in mammalian development occurs during preimplantation development when the outer blastomeres of the embryo form a monolayer of epithelial cells [trophectoderm (TE)] that envelops the remaining blastomeres [inner cell mass (ICM)]. In mice, TE specification occurs in the 8to 16-cell compacted morula. Developmental transition to blastocyst stage is defined by the appearance of a fluid-filled cavity called the blastocoel, which absolutely requires a functional TE (Watson and Barcroft, 2001) (supplementary material Fig. S1). Thus, TE specification is essential for blastocoel formation. Morula-toblastocyst transition is accompanied by a major transition in metabolic energy pathways related to blastocoel formation, concomitant with changes in gene expression that are related to specification of the TE lineage. Identifying the genes that specify the TE and those that regulate changes in metabolism has been the focus of intense, but often separate, investigations. Hence, regulatory genes that directly link preimplantation development to energy homeostasis have yet to be identified. Here, we show that the TEAD4 transcription factor is one such gene. Just prior to blastocoel formation, the embryo switches energy sources abruptly from pyruvate and lactate to glucose, concomitant with a 2.7-fold increase in oxygen consumption (Gardner, 1998; Johnson et al., 2003). This increased oxygen and glucose consumption that results in increased metabolic rate is restricted to the TE where oxidative phosphorylation (OXPHOS) drives the synthesis of ATP (Houghton, 2006; Leese et al., 2008). Na+, K+ATPase within the TE consumes ~60% of this ATP in expanding the blastocoel (Houghton et al., 2003). In fact, an increase in OXPHOS activity with respect to glycolytic activity in developing blastocysts positively correlates with the capacity of the embryo to develop to term following implantation (Gardner, 1998; Gardner, 2008). However, increased energy production via OXPHOS necessarily results in increased production of reactive oxygen species (ROS) (Adelman et al., 1988; Turrens, 1997; Finkel and Holbrook, 2000), which is normally attenuated by antioxidant defense mechanisms present within the embryo and its surroundings (Johnson and Nasr-Esfahani, 1994; Guérin et al., 2001; Orsi and Leese, 2001; Favetta et al., 2007; Betts and Madan, 2008; Kawamura et al., 2010; Zhang et al., 2010). In addition, it is likely that the oxygen level in the female reproductive tract is kept hypoxic relative to the atmospheric oxygen level (3-5% versus 21%) (Gardner and Leese, 1990) in order to minimize ROS production. However, some oxygen is required, because OXPHOS is essential for blastocoel development (Thomson, 1967). Thus, embryos must carefully balance energy production, energy usage and ROS production (termed ‘energy homeostasis’), because failure to do so results in increased oxidative stress, changes in the intracellular redox potential and impaired biosynthetic potential, all of which are detrimental to development (Harvey et al., 2002; Burton et al., 2003; Dumollard et al., 2007; Van Blerkom, 2009). TEAD4 (also known as TEF3) is one of four TEAD proteins in mammals that have nearly identical DNA-binding domains and that bind the same cis-acting sequence (Kaneko and DePamphilis, 1998). They are >80% similar in overall amino acid sequence and bind the same transcriptional co-activators (Vassilev et al., 2001). The Tead4 gene is first expressed at the eight-cell stage, and embryos lacking a functional Tead4 gene arrest development at the morula stage (Nishioka et al., 2009; Nishioka et al., 2008; Yagi et al., 2007). Consequently, Tead4−/− embryos neither form a blastocoel nor implant into the uterus. Moreover, Tead4−/− embryos do not express Cdx2, Gata3 and other genes characteristic of TE. These and other studies reveal that Tead4 acts upstream of genes associated with TE specification and function (Home et al., 2009; Jedrusik et al., 2010; Ralston et al., 2010; Wu et al., 2010). They also do not produce trophoblast stem cells or trophoblast giant cells National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-2753, USA.