Metabolism of Preimplantation Embryo Development: A Bystander or an Active Participant?

Unicellular organisms are exquisitely sensitive to nutrient availability in the environment and have evolved elaborate mechanisms to sense the levels and types of nutrients, altering gene expression patterns accordingly to adjust the metabolic activities required to survive. Thus, environmental cues induce adaptive metabolic differentiation through transcriptional and posttranscriptional changes. Similarly, preimplantation embryos are exposed to various environmental cues within the maternal reproductive tract prior to implantation. Because only "simple" culture conditions are required, it is assumed that these embryos are genetically preprogrammed to develop with little influence from the environment, with the exception of few "necessities" provided by the environment. However, a wealth of literature now suggests that the developing embryos are greatly influenced by the maternal environment. Even though the developing embryos have the capacity and plasticity to deal with nutritional imbalance posed by an altered maternal environment, there is often a trade-off to the overall fitness of those embryos later in life. Despite these studies that underline the general importance of the reproductive environment during development, it is thought that the primary driver of mammalian development is strictly genetic and that metabolic adaptation by the preimplantation embryo is secondary to genetic control. In this review, I propose that not only does the maternal environment of developing preimplantation embryos influence developmental potential, pregnancy outcomes, and postnatal disease states, but that it has an active role in induction and potentiation of the first differentiation event, the production of trophectoderm and inner cell mass lineages.

TEAD/TEF transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein localized in the cytoplasm.

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.

Soggy, a spermatocyte-specific gene, lies 3.8 kb upstream of and antipodal to TEAD-2, a transcription factor expressed at the beginning of mouse development.

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.

Regulation of gene expression at the beginning of mammalian development and the TEAD family of transcription factors.

In mouse development, transcription is first detected in late 1-cell embryos, but translation of newly synthesized transcripts does not begin until the 2-cell stage. Thus, the onset of zygotic gene expression (ZGE) is regulated at the level of both transcription and translation. Chromatin-mediated repression is established after formation of a 2-cell embryo, concurrent with the developmental acquisition of enhancer function. The most effective enhancer in cleavage stage mouse embryos depends on DNA binding sites for TEF-1, the prototype for a family of transcription factors that share the same TEA DNA binding domain. Mice contain at least four, and perhaps five, genes with the same TEA DNA binding domain (mTEAD genes). Since mTEAD-2 is the only one expressed during the first 7 days of mouse development, it is most likely responsible for the TEAD transcription factor activity that first appears at the beginning of ZGE. All four mTEAD genes are expressed at later embryonic stages and in adult tissues; virtually every tissue expresses at least one family member, consistent with a critical role for TEAD proteins in either cell proliferation or differentiation. The 72-amino acid TEA DNA binding domains in mTEAD-2, 3, and 4 are approximately 99% homologous to the same domain in mTEAD-1, and all four proteins bind specifically to the same DNA sequences in vitro with a Kd value of 16-38 nM DNA. Since TEAD proteins appear to be involved in both activation and repression of different genes and do not appear to be functionally redundant, differential activity of TEAD proteins must result either from association with other proteins or from differential sensitivity to chromatin-packaged DNA binding sites.

Transcription factor mTEAD-2 is selectively expressed at the beginning of zygotic gene expression in the mouse.

mTEF-1 is the prototype of a family of mouse transcription factors that share the same TEA DNA binding domain (mTEAD genes) and are widely expressed in adult tissues. At least one member of this family is expressed at the beginning of mouse development, because mTEAD transcription factor activity was not detected in oocytes, but first appeared at the 2-cell stage in development, concomitant with the onset of zygotic gene expression. Since embryos survive until day 11 in the absence of mTEAD-1 (TEF-1), another family member likely accounts for this activity. Screening an EC cell cDNA library yielded mTEAD-1, 2 and 3 genes. RT-PCR detected RNA from all three of these genes in oocytes, but upon fertilization, mTEAD-1 and 3 mRNAs disappeared. mTEAD-2 mRNA, initially present at approx. 5,000 copies per egg, decreased to approx. 2,000 copies in 2-cell embryos before accumulating to approx. 100,000 copies in blastocysts, consistent with degradation of maternal mTEAD mRNAs followed by selective transcription of mTEAD-2 from the zygotic genome. In situ hybridization did not detect mTEAD RNA in oocytes, and only mTEAD-2 was detected in day-7 embryos. Northern analysis detected all three RNAs at varying levels in day-9 embryos and in various adult tissues. A fourth mTEAD gene, recently cloned from a myotube cDNA library, was not detected by RT-PCR in either oocytes or preimplantation embryos. Together, these results reveal that mTEAD-2 is selectively expressed for the first 7 days of embryonic development, and is therefore most likely responsible for the mTEAD transcription factor activity that appears upon zygotic gene activation.

Regulation of gene expression at the beginning of mammalian development.

The maternal to zygotic transition can be viewed as a cascade of events that begins when fertilization triggers the zygotic clock that delays early ZGA until formation of a 2-cell embryo. Early ZGA, in turn, appears to be required for expression of late ZGA, and late ZGA is required to form a 4-cell embryo. ZGA in mammals is a time-dependent mechanism rather than a cell cycle-dependent mechanism that delays both transcription and translation of nascent transcripts. Thus, zygotic gene transcripts appear to be handled differently than maternal mRNA, a phenomenon also observed in Xenopus (55). The length of this delay is species-dependent, occurring at the 2-cell stage in mice, the 4-8-cell stage in cows and humans, and the 8-16-cell stage in sheep and rabbits (4). However, concurrent with formation of a 2-cell embryo in the mouse and rabbit (47,56), perhaps in all mammals, a general chromatin-mediated repression of promoter activity appears. Repression factors are inherited by the maternal pronucleus from the oocyte but are absent in the paternal pronucleus and not available until sometime during the transition from a late 1-cell to a 2-cell embryo. This means that paternally inherited genes are exposed to a different environment in fertilized eggs than are maternally inherited genes, a situation that could contribute to genomic imprinting. Chromatin-mediated repression of promoter activity prior to ZGA is similar to what is observed during Xenopus embryogenesis (31,32) and ensures that genes are not expressed until the appropriate time in development when positive acting factors, such as enhancers, can relieve this repression. The ability to use enhancers appears to depend on the acquisition of specific co-activators at the 2-cell stage in mice and perhaps later in other mammals (47,56), concurrent with ZGA. Even then, the mechanism by which enhancers communicate with promoters changes during development (Fig. 2), providing an opportunity for enhancer-mediated stimulating of TATA-less promoters (e.g. housekeeping genes) early during development while eliminating this mechanism later during development.(ABSTRACT TRUNCATED AT 400 WORDS)

Activation of the silent progesterone receptor gene by ectopic expression of estrogen receptors in a rat fibroblast cell line.

We describe the construction and characterization of a novel estrogen (E2)-responsive cell line, Rat1+ER, which ectopically expresses estrogen receptor (ER). Human ER cDNA was introduced by retrovirus-mediated gene transfer into the Rat1 cell line, which does not express functional ER endogenously. Rat1+ER cells express functional ER based on radioreceptor assays, immunoblotting, and transient transfection experiments using E2-responsive reporter plasmids. The effects of this ectopic ER expression were studied on three endogenous E2-responsive genes, prolactin (PRL), progesterone receptor (PR), and epidermal growth factor receptor (EGFR). PRL, usually expressed in the lactotrophs of the pituitary, is not expressed at all in Rat1+ER cells, with or without E2 addition, and appears to require other factors for expression. In contrast, although PR is not expressed in Rat1 cells, it is induced in Rat1+ER cells upon the addition of E2. This induction appears to occur at the transcriptional level and is insensitive to cycloheximide treatment. This is one of the few examples where the expression of one gene activates an otherwise silent gene. Another contrasting observation is that, although EGFR is basally expressed in Rat1+ER cells, the addition of E2 has no effect. Our studies paint a complicated picture of E2 regulation of endogenous genes: the activation of the PR gene may only require the presence of E2 and ER, whereas EGFR and PRL genes require factors in addition to ER for basal as well as E2-regulated expression.

Involvement of the coding sequence for the estrogen receptor gene in autologous ligand-dependent down-regulation.

The estrogen receptor (ER) appears to be down-regulated by its own ligand in some estrogen (E2)-responsive tissues as well as in cell lines such as MCF-7 and GH3. Surprisingly, we observed ER down-regulation in a newly constructed E2-responsive cell line (Rat1 + ER), in which expression of the coding region of the ER cDNA was driven by the Moloney murine leukemia virus long terminal repeat. We present evidence that the coding region of the ER cDNA, but not the Moloney murine leukemia virus long terminal repeat, possesses a sequence(s) necessary for ER down-regulation. The observed down-regulation occurs at ligand-binding and protein, as well as mRNA, levels. Marked decreases in both protein and mRNA levels were observed as early as 3 h after E2 treatment. Furthermore, maximal down-regulation occurred by 18-24 h with ligand-binding, and mRNA levels reached approximately 20% that of controls. ER down-regulation in Rat1 + ER cells is only partially inhibited by the presence of cycloheximide and therefore suggests a direct participation of the ER in this process. E2 does not appear to influence the stability of the ER transcript, which implies that negative regulation is occurring at the transcriptional level. Finally, since we can demonstrate ER binding to a portion of the cDNA sequence, we propose a mechanism whereby ER binding to its putative negative element leads to transcriptional repression of the upstream promoter.

Role of conserved sequence elements 9L and 2 in self-splicing of the Tetrahymena ribosomal RNA precursor.

Oligonucleotide-directed mutagenesis has been used to alter highly conserved sequences within the intervening sequence (IVS) of the Tetrahymena large ribosomal RNA precursor. Mutations within either sequence element 9L or element 2 eliminate splicing activity under standard in vitro splicing conditions. A double mutant with compensatory base changes in elements 9L and 2 has accurate splicing activity restored. Thus, the targeted nucleotides of elements 9L and 2 base-pair with one another in the IVS RNA, and pairing is important for self-splicing. Mutant splicing activities are restored by increased magnesium ion concentrations, supporting the conclusion that the role of the targeted bases in splicing is primarily structural. Based on the temperature dependence, we propose that a conformational switch involving pairing and unpairing of elements 9L and 2 is required for splicing.