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.

One goal of developmental biology is to identify mechanisms that regulate the onset of zygotic gene transcription and initiate the developmental program. In the mouse, DNA transcription stops when oocytes undergo meiotic maturation to form eggs and does not begin again until the late 1-cell stage. However, transcription does not appear to be coupled to translation until the 2-cell stage (Nothias et al., 1996) where synthesis of zygotic proteins begins (Latham et al., 1991). This delay in expression of zygotic genes is regulated by a time-dependent mechanism about which little is known (Nothias et al., 1995). One component may be the appearance of an enhancer specific cofactor in 2-cell embryos that is absent in oocytes and 1-cell embryos (Majumder et al., 1997). Another may be the appearance of specific transcription factors that regulate the expression of other zygotic genes. Recent studies revealed that protein synthesis is required for embryonic genome activation, consistent with a need to synthesize essential transcription factors lacking in the oocyte (Wang and Latham, 1997). Only a few transcription factors have so far been identified and extensively studied in mouse oocytes and preimplantation embryos (Schultz, 1993; Majumder and DePamphilis, 1995; Nothias et al., 1995).

One approach to investigating the regulation of gene expression at the beginning of mammalian development has been to inject plasmid encoded reporter genes into the nuclei of mouse oocytes and preimplantation embryos (Majumder et al., 1993; Majumder and DePamphilis, 1995; Nothias et al., 1995). The injected DNA can replicate or express an encoded reporter gene only when specific cis-acting regulatory sequences are present and they are provided with their cognate trans-acting proteins. Moreover, replication and expression of plasmid encoded genes occur only when the embryonic genome executes the same functions during its normal developmental program. These studies indicate that the response of injected plasmids reflects physiological controls that govern expression of cellular genes, thereby revealing the embryo’s capacity for DNA replication and gene expression, as well as its requirements for specific regulatory elements.

This microinjection strategy has been used to search for tran-scription enhancers that function in cleavage stage mouse embryos and during the initial activation of zygotic gene expression (Martinez-Salas et al., 1989; Melin et al., 1993). A survey of polyomavirus mutants that replicate in undifferentiated mouse embryonal carcinoma or embryonic stem cells revealed a single point mutation common to enhancers that also functioned in cleavage stage embryos. This mutation created a binding site for Transcription Enhancer Factor-1 (TEF-1; Xiao et al., 1991). This DNA binding site can stimulate transcription when present in either promoters (Farrance et al., 1992) or enhancers (Melin et al., 1993). The most effective enhancers consist of two tandem TEF-1 DNA binding sites separated by about 53 bp. Stimulation by the enhancer from polyomavirus F101, for example, ranges from 20 to >300-fold, depending on the promoter tested and the amount of DNA injected (Majumder et al., 1993). Since the activity of the F101 enhancer in early embryos can be duplicated by engineering a series of tandem TEF-1 DNA binding sites, a transcription factor must be present at the onset of zygotic gene expression (ZGE) that can utilize this DNA binding site. However, the obvious candidate for this role, mouse TEF-1, does not appear suitable, because embryos homozygous for a disruption in the TEF-1 gene survive past the preimplantation stage (Chen et al., 1994). Therefore, either TEF-1 activity is present in preimplantation embryos but its function is not required, or another related gene can mediate expression of endogenous target genes.

The TEF-1 transcription factor contains a highly conserved 72 amino acid DNA binding domain (TEA domain) that is found in transcription factors from widely divergent species (Burglin, 1991; Blatt and DePamphilis, 1993). TEF-1 mRNA has been detected in many adult mouse tissues (Blatt and DePamphilis, 1993), and TEF-1 protein has been associated with the regulation of a variety of cell-type specific genes (Yockey et al., 1996; ref. therein). More recently, three additional mouse genes have been identified that share the same TEA domain as mouse TEF-1 and bind to the TEF-1 DNA sequence motif in vitro (see Results). Therefore, in order to distinguish this family of genes from unrelated factors that fortuitously bear the same acronym, and to recognize the single common denominator among these proteins, we refer to this gene family as murine TEA Domain genes (mTEAD-1 to 4). Here we report that mTEAD transcription factor activity was not detected in oocytes, but first appeared at the 2-cell stage in mouse development, concomitant with the onset of zygotic gene expression. mTEAD-2 was the principal member of the mTEAD transcription factor gene family that was expressed from cleavage stage preimplantation embryos up to day-7 embryos. Thus, mTEAD-2 is one of the first transcription factors produced at the beginning of mouse development where its presence most likely accounts for the TEAD-dependent enhancer activity observed at the onset of zygotic gene expression.

Embryo and oocyte isolation, culture, and microinjection

All procedures were performed with CD-1 mice (Charles River) and have been described previously (DePamphilis et al., 1988; Miranda et al., 1993; Hogan et al., 1994). Germinal vesicle-stage oocytes isolated from 14-day-old mice were used for oocyte microinjection experiments. The day the vaginal plug was observed was designated as day 1. For quantitative RT-PCR analyses, oocytes and embryos from B6D2 F1 mice were used as described by Rambhatla et al. (1995).

Plasmids

pluc, pF101tkluc, pS6Tluc have been described by Martinez-Salas et al. (1989); Majumder et al. (1993). pGT4Tluc consisted of four tandem copies of the GTIIC site 30-mer containing the TEF-1 DNA binding sites found in the polyomavirus F101 enhancer (Melin et al., 1993). This sequence was constructed by ligating together two oligomers. The first oligomer contained a 5′ restriction site for BglII and a 3′ AGCC overhang, whereas the second oligomer contained a 5′ TCGG overhang and a 3′ restriction site for EcoRI. After annealing and phosphorylating the complementary strands, the two fragments were allowed to anneal and ligated into the EcoRI/BglII sites of p2025 upstream and proximal to a TATA box (Majumder et al., 1993). A fragment containing the 4 TEF-1 sites and a TATA box was amplified by PCR using SP6 and T7 primers with a 5′-SacI restriction site. Since this fragment contained a SacI restriction site next to the SP6 primer, the fragment was digested with SacI and ligated into the SacI site of pluc.

cDNA library screening

A PCC4 cDNA library (Stratagene; Blatt and DePamphilis, 1993) was screened under low stringency conditions (Giguere et al., 1987), using a mouse TEA domain probe from mTEAD-1. This probe (220 nucleotides) was synthesized by PCR using primers A and B (Table 1) which flank the TEA domain of mTEAD-1 cDNA (Blatt and DePamphilis, 1993). Probe was hybridized with nitrocellulose filters in 6× SSPE, 5× Denhardt’s, 0.1% sodium dodecylsulfate (SDS), 1 mM sodium pyrophosphate, 100 μg/ml sheared salmon sperm DNA, and 25% v/v formamide at 42°C overnight. The filters were washed three times in 2× SSC, 0.1% SDS for 20 minutes at 55°C. The positive phagemids were converted to pBluescript plasmids (Stratagene). These plasmids were sequenced from both ends by DyeDeoxy Sequencing (Applied Biosystems). The 5′ end of mTEAD-2 and the 3′ end of mTEAD-3 were isolated by rapid amplification of cDNA ends (5′/3′ RACE) method (Frohman et al., 1988) from heart and lung tissues, respectively. The cDNA sequences for both mTEAD-2 and -3 have been submitted to GenBank (accession numbers, Y10026 and Y10027, respectively).

Table 1.

Oligonucleotides used as primers and probes

Oligonucleotides used as primers and probes
Oligonucleotides used as primers and probes

Northern blotting-hybridization assays

mTEAD-1 probe was identical to the TEA domain probe used for library screening. For mTEAD-2 and 3 probes (approx. 1.8 kb and approx. 2.0 kb, respectively), both cDNAs were released from the pBluescript vector by digesting with EcoRI. Human β-actin cDNA was obtained from Clontech. The probes were radiolabeled with [α- 32P]dCTP using a Random Primed DNA labeling kit (Boehringer Mannheim). Hybridizations were carried out at 42°C in solution containing 5× SSPE, 10× Denhardt’s solution, 1 mM sodium pyrophosphate, 50% formamide, 100 μg/ml sheared, denatured salmon sperm DNA, and 2% SDS. The blots were washed at high stringency (50°C, 0.1× SSC), exposed for autoradiography, stripped by incubating the blots in boiling 0.1× SSPE, 0.5% SDS and rehybridized.

In situ hybridization

In situ hybridizations with digoxigenin-labeled probes were carried out on 10 μm cryostat sections as described by Schaeren-Wiemers and Gerefin-Moser (1993). Eight to twelve decidua (isolated from day-7 pregnant females) or ovaries were dissected in PBS, placed directly in OCT Tissue tek (Baxter) and frozen on dry ice prior to sectioning. Probes for in situ hybridization were synthesized as follows. C-terminal half, which does not contain the TEA domain, of the three gene family members, were synthesized by PCR using Pfu DNA polymerase (Stratagene) and specific primers for mTEAD-1 (C,D), 2 (E,F), and 3 (G,H). The PCR products were cloned into pCRII (Invitrogen) and the EcoRI digested inserts were cloned into Bluescript. T7 and T3 RNA polymerases (Promega) were used to generate antisense and sense probes for mTEAD-1, 2, and 3.

RT-PCR

Oocytes and embryos (100/assay) were isolated as described previously (DePamphilis et al., 1988), frozen in an ethanol/dry ice bath, and stored at −80°C. After addition of 10 μg rRNA (Boehringer Mannheim), 200 μl of denaturing solution was used to lyse cells and extract RNA using RNAgents Total RNA Isolation System (Promega). RNA was resuspended in 10 μl of DEPC H2O and 1 μl was used to quantify RNA recovery by spectrophotometry. Reverse transcription was performed with the remaining RNA using random primer, RNAse inhibitor, and Moloney murine leukemia virus reverse transcriptase (Perkin Elmer). 50 ng of total RNA from mouse lung, F9 and MPC11 cell lines were used as controls. Aliquots (5 μl) of the same RT reaction (20 μl) were used for the four separate PCR reactions. Specific primers used for mTEAD-1, 2, 3, and 4 were I and J, K and L, M and N, and O and P, respectively (Table 1). These primers did not generate a specific product of the correct size when 500 ng of F9 genomic DNA were used as a template, presumably due to presence of introns between the primers. Therefore, any contaminating genomic DNA would not contribute to the specific amplified products observed in the assay. Except for primer K, all primers flank the TEA DNA binding domain. Although primer K is within the TEA DNA binding domain, it, along with primer L, did not produce specific products from plasmids containing mTEAD-1, 3, and 4 cDNAs. The primers for TEAD-4 were designed so that two alternatively spliced RNAs of different sizes could be amplified simultaneously. PCR reactions (100 μl) were carried out with Taq DNA polymerase (Perkin Elmer) for 40 cycles. PCR products (16 μl) were fractionated by 2% agarose electrophoresis, stained with ethidium bromide, denatured and transferred to Zeta Probe GT membrane (Bio-Rad). The membrane was hybridized with 5′-end labeled 32P-oligonucleotides internal to the amplicon. Oligonucleotide probes for mTEAD-1, -2, -3, -4 were Q, R, S, and T, respectively.

Quantitative RT-PCR was done as described by Rambhatla et al. (1995). The mTEAD-2 probe used for hybridization was a 650 bp XhoI/DraI DNA fragment. The probe used for mTEAD-3 was an approx. 220 bp EcoRI fragment from pCRII vector containing the 3′-end of TEAD-3 cDNA obtained by 3′ RACE (see above). Both of these probes contained putative polyadenylation signals. The probe used for TEAD-1 was approx. 250 bp HindIII-digested fragment of mTEAD-1 (TEF-1) clone 13-1 (Blatt and DePamphilis, 1993).

mTEAD transcription factor activity first appeared when zygotic gene expression began

To determine when functional mTEAD transcription factor is produced at the beginning of mouse development, a plasmid encoded reporter gene whose activity depended on mTEAD was microinjected into the nuclei of oocytes, 1-cell embryos, or 2-cell embryos under conditions that produce the greatest amount of transient gene expression (Miranda et al., 1993). To confirm that mTEAD-dependent enhancer activity was present in cleavage-stage embryos, two plasmids (Melin et al., 1993) were microinjected individually into 2-cell embryos. One plasmid contained a mTEAD-dependent enhancer consisting of four tandem copies of the mTEAD binding site found in polyomavirus F9-1 upstream of the polyomavirus early gene promoter linked to the firefly luciferase reporter gene [p(GT30)4-Pyluc]. A second plasmid [p(wt30)4-Pyluc] was identical to the first except for a single base pair change that eliminates binding of mTEAD-1, 2 and 3 proteins in vitro (data not shown; Davidson et al., 1988). This was consistent with the effects of similar mutations in the functionally equivalent SV40 GTIIC site on binding of mTEAD-1, 2, 3 and 4 proteins (Xiao et al., 1991; Yasunami et al., 1995; Jacquemin et al., 1996; Yasunami et al., 1996). The results (Fig. 1A) showed that a mTEAD-dependent enhancer could strongly (>500-fold) stimulate promoter activity in early cleavage embryos.

Fig. 1.

mTEAD transcription factor activity at the beginning of mouse development. (A) Plasmids encoding the luciferase gene driven by a polyoma virus promoter linked to enhancers consisting of either four tandem F9-1 mutation (TEF-1 DNA binding sites; GT4- Pyluc, black box) or four tandem wild-type sequences (wt4-Pyluc, white box) were injected into one of the nuclei of 2-cell embryos. (B,C) Plasmids encoding the luciferase gene driven either by four tandem TEF-1 DNA binding sites adjacent to a TATA box (pGT4Tluc, black boxes, B), or the HSV thymidine kinase promoter (ptkluc; narrow hatched boxes, C), or six tandem Sp1 DNA binding sites adjacent to a TATA-box (pS6Tluc, broad hatched boxes, C) were injected individually into the germinal vesicle (nucleus) of oocytes, the paternal pronucleus of 1-cell embryos, and one of the two zygotic nuclei of 2-cell embryos as described by DePamphilis et al. (1988). Oocytes were cultured in the presence of dibutyryl cAMP to prevent germinal vesicle breakdown and cessation of transcription. Early 1-cell embryos were cultured in the presence of aphidicolin (a specific inhibitor of replicative DNA polymerases) in order to arrest their development as they entered S-phase and prevent repression of transcription during formation of a 2-cell embryo. Two-cell embryos were isolated from pregnant females, and then injected and cultured under the same conditions as 1-cell embryos. Since most of these embryos had already completed S-phase, they soon cleaved into 4-cell embryos where they arrested their development as they entered S-phase. Plasmid DNA concentrations were 0.5 mg/ml (oocytes), and 0.15 mg/ml (1-cell and 2-cell embryos). Injected embryos were assayed for luciferase activity at 24 to 44 hours post-injection. Each data point is the mean from 44 to 167 injected oocytes or embryos. These levels of activity have been corrected for background levels produced by a promoterless plasmid (pluc). Error bars indicate the standard error of the mean. The ratio of activities produced by the indicated plasmids are shown in D.

Fig. 1.

mTEAD transcription factor activity at the beginning of mouse development. (A) Plasmids encoding the luciferase gene driven by a polyoma virus promoter linked to enhancers consisting of either four tandem F9-1 mutation (TEF-1 DNA binding sites; GT4- Pyluc, black box) or four tandem wild-type sequences (wt4-Pyluc, white box) were injected into one of the nuclei of 2-cell embryos. (B,C) Plasmids encoding the luciferase gene driven either by four tandem TEF-1 DNA binding sites adjacent to a TATA box (pGT4Tluc, black boxes, B), or the HSV thymidine kinase promoter (ptkluc; narrow hatched boxes, C), or six tandem Sp1 DNA binding sites adjacent to a TATA-box (pS6Tluc, broad hatched boxes, C) were injected individually into the germinal vesicle (nucleus) of oocytes, the paternal pronucleus of 1-cell embryos, and one of the two zygotic nuclei of 2-cell embryos as described by DePamphilis et al. (1988). Oocytes were cultured in the presence of dibutyryl cAMP to prevent germinal vesicle breakdown and cessation of transcription. Early 1-cell embryos were cultured in the presence of aphidicolin (a specific inhibitor of replicative DNA polymerases) in order to arrest their development as they entered S-phase and prevent repression of transcription during formation of a 2-cell embryo. Two-cell embryos were isolated from pregnant females, and then injected and cultured under the same conditions as 1-cell embryos. Since most of these embryos had already completed S-phase, they soon cleaved into 4-cell embryos where they arrested their development as they entered S-phase. Plasmid DNA concentrations were 0.5 mg/ml (oocytes), and 0.15 mg/ml (1-cell and 2-cell embryos). Injected embryos were assayed for luciferase activity at 24 to 44 hours post-injection. Each data point is the mean from 44 to 167 injected oocytes or embryos. These levels of activity have been corrected for background levels produced by a promoterless plasmid (pluc). Error bars indicate the standard error of the mean. The ratio of activities produced by the indicated plasmids are shown in D.

The ability of a mTEAD-dependent enhancer to stimulate promoter activity could not be used to assay mTEAD transcription factor activity in oocytes or 1-cell embryos, because enhancers cannot be utilized in mouse cells until formation of a 2-cell embryo (Majumder et al., 1997). However, the same transcription factors can be assayed prior to formation of a 2-cell embryo by placing their sequence-specific DNA binding sites proximal, rather than distal, to the mRNA start site (Majumder et al., 1993, 1997). Previous studies (Majumder et al., 1993) showed that promoter activity depended strictly on the presence of both specific transcription factors and their cognate DNA binding sites. These genes are most active when injected into the paternal pronucleus of S-phase arrested 1-cell embryos where promoters are not repressed (Martinez-Salas et al., 1989; Wiekowski et al., 1991, 1993; Majumder et al., 1993, 1997), and least active in oocytes and 2-cell to 4-cell embryos where chromatin structure mediates repression of promoter activity (Henery et al., 1995; Wiekowski et al., 1997). Furthermore, in fertilized mouse eggs, the onset of transcription and translation of both the zygotic genome as well as injected plasmids is delayed by a time dependent mechanism until the 2-cell stage in development (Nothias et al., 1995, 1996). Thus, even when morphological development stops as 1-cell embryos are arrested at the beginning of S-phase, ZGE still begins at the normal time (2-cell stage) after fertilization. Therefore, the activity of promoters injected into the paternal pronucleus of S-phase arrested 1-cell embryos reflected the amount of cognate transcription factor activity present at the onset of ZGE, in the absence of chromatin mediated repression.

To address whether mTEAD activity can be detected prior to the formation of 2-cell embryos, a mTEAD-dependent promoter consisting of four tandem mTEAD binding sites was placed 10 bp upstream of a TATA box (pGT4Tluc). For comparison, two Sp1-dependent promoters were also examined: ptkluc contained the herpes simplex virus thymidine kinase promoter, consisting of two Sp1 sites, a CTF site, and a TATA box; pS6Tluc contained a tandem series of six Sp1 DNA binding sites and a TATA box. As expected, oocytes and 1-cell embryos were capable of transcribing control reporter genes driven by Sp1-dependent promoters (Fig. 1C) since both Sp1 and TATA binding proteins have been shown to be present in oocytes, fertilized eggs and early cleavage stage embryos (Majumder et al., 1993; Worrad et al., 1994).

In contrast, mTEAD-dependent promoter (pGT4Tluc; Fig. 1B) was inactive in oocytes and became active in S-phase arrested 1-cell embryos (chronologically at 2-cell stage) and in 2/4-cell embryos (chronologically at 4/8-cell stage). Since oocytes were capable of utilizing the Sp1-dependent promoters (Fig. 1C) but not the mTEAD-dependent promoter (Fig. 1B), mTEAD transcription factor activity must be absent in oocytes. mTEAD-dependent promoter activity was detected in S-phase arrested 1-cell embryos but was only about 10% as active as Sp1-dependent promoters (Fig. 1D). However, by the time 2-cell and 4-cell embryos had formed, when all promoters are subjected to chromatin-mediated repression, all three promoters produced comparable amounts of luciferase, suggesting that the levels of active Sp1 and mTEAD proteins were also comparable (Fig. 1D).

These data demonstrated that mTEAD activity first appears at the onset of ZGE. In S-phase arrested 1-cell embryos, ZGE begins about 12 hours after injection, concurrent with the beginning of a shutdown of maternal mRNA translation (Wiekowski et al., 1991; Nothias et al., 1995). Thus, less time was available for assembly of an active mTEAD-dependent promoter than for an active Sp1-dependent promoter, because Sp1 protein was inherited from the oocyte and therefore was present when the plasmid gene was injected. However, when the same plasmids were injected into late 2-cell embryos isolated from pregnant females, both mTEAD and Sp1 transcription factor activities were already present, because ZGE begins immediately after 1-cell embryos cleave into 2-cell embryos (Nothias et al., 1996), approximately 12 hours prior to injection. The data obtained from both mTEAD-dependent enhancer/promoter constructs show that mTEAD activity first appears at a time chronologically equivalent to the 2-cell stage, as reflected in the S-phase arrested embryos, and then continues to be expressed into the 4-cell stage.

Only mTEAD-2 RNA was detected during preimplantation development

The above results demonstrated that mTEAD transcription factor activity first appeared at the 2-cell stage in mouse development. However, mouse embryos disrupted in the mTEAD-1 (TEF-1) locus can develop past the preimplantation stage (Chen et al., 1994), suggesting that another gene, related to mTEAD-1, mediates the mTEAD-dependent enhancer/promoter function in preimplantation embryos. Since this putative mTEAD-1 like protein must be able to bind to the mTEAD DNA binding sequence, it presumably contains a TEA DNA binding domain. Therefore, we screened an embryonic carcinoma cell line with the TEA DNA binding domain probe from mTEAD-1 under low stringency conditions to identify additional mTEAD genes. In this way, mTEAD-2 and 3 cDNAs were isolated (GenBank accession numbers, Y10026 and Y10027, respectively). mTEAD-2 is identical to ETF (Yasunami et al., 1995) and TEF-4 (Jacquemin et al., 1996), while mTEAD-3 is identical to ETFR-1 (Yasunami et al., 1996). A fourth member of this mouse gene family (designated mTEAD-4) has recently been isolated (TEFR-1; Yockey et al., 1996). All four proteins have nearly identical DNA binding domains and can bind specifically to the SV40 GTIIC and polyomavirus F9-1 sequences (see above). Southern analyses of genomic DNA restriction endonuclease fragments under low stringency conditions were consistent with the presence of only four mTEAD genes (data not shown).

To determine whether mTEAD genes were expressed as maternal mRNAs, mTEAD-1, 2, and 3 RNAs were assayed in oocytes using in situ hybridization. Frozen tissue sections through adult mouse ovaries were hybridized with digoxigenin-labeled RNA probes complementary to the C-terminal portion of the mTEAD-1, 2, and 3 cDNA. A specific mTEAD mRNA was considered present in cells only if the antisense probe produced a signal significantly greater than that produced by the sense probe. These analyses showed that mTEAD-2 RNA was not detected in oocytes using an antisense probe, even though the signal was clearly present in the granulosa cells within the follicles (Fig. 2A,C). Neither the antisense probes for mTEAD-1 and 3, nor the three sense probes hybridized with any of the cells in the ovary (Fig. 2B,D; data not shown). mTEAD-4 RNA was not assayed in this and subsequent studies, because it was not detected in oocytes or preimplantation embryos by RT-PCR (Fig. 3). Therefore, mTEAD-1, 2 and 3 were not expressed in oocytes at levels detectable by in situ hybridization.

Fig. 2.

Distribution of mTEAD-2 mRNA in the adult mouse ovary. In situ hybridization was carried out as described in Materials and methods. Adjacent serial sections through the ovary were hybridized with antisense (A,C) or sense (B,D) mTEAD-2 probes. C and D show higher magnifications (6.25×) of one of the follicles from A and B, respectively. No specific staining was detected in adjacent sections when hybridized with either mTEAD-1 or mTEAD-3 specific probes (data not shown). Some follicles (Fc) are indicated in A, whereas the oocyte (Oc), granulosa cells (Gc), and theca cells (Tc) are indicated in C. Bar represents 50 μm.

Fig. 2.

Distribution of mTEAD-2 mRNA in the adult mouse ovary. In situ hybridization was carried out as described in Materials and methods. Adjacent serial sections through the ovary were hybridized with antisense (A,C) or sense (B,D) mTEAD-2 probes. C and D show higher magnifications (6.25×) of one of the follicles from A and B, respectively. No specific staining was detected in adjacent sections when hybridized with either mTEAD-1 or mTEAD-3 specific probes (data not shown). Some follicles (Fc) are indicated in A, whereas the oocyte (Oc), granulosa cells (Gc), and theca cells (Tc) are indicated in C. Bar represents 50 μm.

Fig. 3.

Expression of mTEAD-1, 2, 3, and 4 RNA in oocytes and preimplantation embryos. Total RNA from 100 each of oocytes, 1-cell embryos, 2-cell embryos, morula, and blastocysts was reverse transcribed using random primers and then aliquots from the same reverse transcription reaction were amplified by PCR with primers specific for mTEAD-1, 2, 3, and 4 (TEFR1) cDNAs. The amplified products were then fractionated by agarose gel electrophoresis, stained with ethidium bromide (top panel in each set), and detected by Southern hybridization using specific 32P-labeled oligonucleotides internal to the PCR primers. RNA (50 ng/lane) from mouse lung, F9 cells and MPC11 cells were also simultaneously analyzed as controls.

Fig. 3.

Expression of mTEAD-1, 2, 3, and 4 RNA in oocytes and preimplantation embryos. Total RNA from 100 each of oocytes, 1-cell embryos, 2-cell embryos, morula, and blastocysts was reverse transcribed using random primers and then aliquots from the same reverse transcription reaction were amplified by PCR with primers specific for mTEAD-1, 2, 3, and 4 (TEFR1) cDNAs. The amplified products were then fractionated by agarose gel electrophoresis, stained with ethidium bromide (top panel in each set), and detected by Southern hybridization using specific 32P-labeled oligonucleotides internal to the PCR primers. RNA (50 ng/lane) from mouse lung, F9 cells and MPC11 cells were also simultaneously analyzed as controls.

To determine which mTEAD gene might be responsible for TEAD activity in preimplantation embryos, a more sensitive assay was employed using reverse transcriptase coupled with the polymerase chain reaction (RT-PCR). mTEAD RNAs were assayed in oocytes, 1-cell embryos, 2-cell embryos, morula and blastocysts. Mouse lung and teratocarcinoma F9 cells provided positive controls, while mouse lymphoma cell line MPC11, in which TEAD-1 RNA has been reported absent (Xiao et al., 1991), provided a negative control. cDNA was first synthesized using reverse transcriptase (RT) and random primers. Aliquots of each RT reaction were then amplified using primers specific for one of the four mTEAD genes (see Materials and methods). The resulting amplified DNA sequences were visualized by ethidium bromide staining and by Southern blotting hybridization using probes internal to the two PCR primers (Fig. 3).

All four mTEAD RNAs were detected in lung and F9 cells, while only low levels of mTEAD-3 and 4, and no mTEAD-1 or 2 RNA was detected in MPC11 cells. In each case, amplified DNA products of the expected sizes were produced, and these products were present in some cells but not in others, confirming the specificity and the validity of the RT-PCR assay. Only transcripts from mTEAD-1, 2 and 3 were detected in oocytes. Following fertilization, mTEAD-1 and 3 RNA disappeared, whereas the amount of mTEAD-2 RNA steadily increased during preimplantation development, reaching levels in morula and blastocysts great enough to be detected by ethidium bromide staining. These results suggest that mTEAD-2 is the principal mTEAD gene family member expressed during preimplantation development.

To provide a more quantitative assessment of mTEAD-2 expression, the number of copies of mTEAD-2 mRNA was estimated using a RT-PCR assay based on uniform amplification of the 3′-terminal region of all poly(A)+ mRNAs (Rambhatla et al., 1995). The amplified products could then be quantified by hybridization with 32P-labeled DNA probes specific for each mTEAD gene. The data were expressed as cpm bound per ovum or embryo (Fig. 4A) and used to calculate the number of mRNA copies per ovum or embryo (Fig. 4B; Rambhatla et al., 1995).

Fig. 4.

Quantitation of mTEAD-2 mRNA from oocytes to blastocysts. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the entire population of poly(A)+ mRNA from mouse ova and embryos while preserving the relative abundance of each mRNA in the cDNA population (Rambhatla et al., 1995). Between 3 to 8 samples were used per stage, and the error bars denote standard errors. (A) 32P-labeled DNA probe specific for mTEAD-2 was hybridized with this cDNA population and the cpm/ovum or embryo recorded. cpm were 268±40 in oocytes, 743±259 in 2-cell embryos, and 3103±347 in blastocysts. (B) The data in panel A were used to calculate the number of mTEAD-2 mRNA copies as described (Rambhatla et al., 1995). (C) The scale used in panel B was expanded to show the change in mTEAD-2 mRNA following fertilization. One-cell embryos were isolated from pregnant females and cultured in vitro to allow development up to the blastocyst stage (white squares). Some 1-cell embryos were cultured in the presence of α-amanitin to prevent transcription (black circles). Some 2-cell embryos were isolated from pregnant females (black squares). A curve (4th order polynomial) was fitted to the 17 points post-hCG in each panel and then shaded to reveal changes in the amount of mRNA.

Fig. 4.

Quantitation of mTEAD-2 mRNA from oocytes to blastocysts. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the entire population of poly(A)+ mRNA from mouse ova and embryos while preserving the relative abundance of each mRNA in the cDNA population (Rambhatla et al., 1995). Between 3 to 8 samples were used per stage, and the error bars denote standard errors. (A) 32P-labeled DNA probe specific for mTEAD-2 was hybridized with this cDNA population and the cpm/ovum or embryo recorded. cpm were 268±40 in oocytes, 743±259 in 2-cell embryos, and 3103±347 in blastocysts. (B) The data in panel A were used to calculate the number of mTEAD-2 mRNA copies as described (Rambhatla et al., 1995). (C) The scale used in panel B was expanded to show the change in mTEAD-2 mRNA following fertilization. One-cell embryos were isolated from pregnant females and cultured in vitro to allow development up to the blastocyst stage (white squares). Some 1-cell embryos were cultured in the presence of α-amanitin to prevent transcription (black circles). Some 2-cell embryos were isolated from pregnant females (black squares). A curve (4th order polynomial) was fitted to the 17 points post-hCG in each panel and then shaded to reveal changes in the amount of mRNA.

mTEAD-2 poly(A)+ mRNA was present in oocytes and unfertilized eggs at 4000 to 5000 copies per cell. Following fertilization, the level of mTEAD-2 mRNA decreased until the late 2/4-cell stage and then increased rapidly (Fig. 4C). Since mTEAD-2 mRNA levels in 1-cell and 2-cell embryos were insensitive to α-amanitin (a specific inhibitor of RNA polymerase II), most of this mRNA was inherited from the egg. From the 8-cell to the blastocyst stage, mTEAD-2 mRNA accumulated dramatically, consistent with its expression from zygotic genes. In blastocysts, the level of mTEAD-2 mRNA was about 100,000 copies per embryo, or about 15% the level of β-actin mRNA in blastocysts (Rambhatla et al., 1995). Thus, the level of mTEAD-2 in blastocysts was about 20-fold greater than in oocytes or about 50-fold greater than in 2-cell and 4-cell embryos.

In striking contrast to mTEAD-2 mRNA, the level of mTEAD-3 poly(A)+ mRNA in ova and preimplantation embryos was essentially indistinguishable from background (Fig. 5). A similar result was obtained with mTEAD-1.

Fig. 5.

Quantitation of mTEAD-1 and mTEAD-3 mRNA from oocytes to blastocysts. The experiment described in Fig. 4 was repeated using 32P-labeled DNA probes specific for either mTEAD-1 or mTEAD-3. These data were then compared with those for mTEAD-2 (shaded area is from Fig. 3C). cpm for mTEAD-1 were 37±4 in oocytes, 58±7 in 2-cell embryos and 67±4 in blastocysts. cpm for mTEAD-3 were 181±14 in oocytes, 89±28 in 2-cell embryos and 149±17 in blastocysts.

Fig. 5.

Quantitation of mTEAD-1 and mTEAD-3 mRNA from oocytes to blastocysts. The experiment described in Fig. 4 was repeated using 32P-labeled DNA probes specific for either mTEAD-1 or mTEAD-3. These data were then compared with those for mTEAD-2 (shaded area is from Fig. 3C). cpm for mTEAD-1 were 37±4 in oocytes, 58±7 in 2-cell embryos and 67±4 in blastocysts. cpm for mTEAD-3 were 181±14 in oocytes, 89±28 in 2-cell embryos and 149±17 in blastocysts.

However, since the 3′-ends of all mTEAD-1 clones reported do not contain a recognizable polyadenylation signal (Blatt and DePamphilis, 1993; Shimizu et al., 1993), our 3′-probe may not have been close enough to the polyA tail to register in this assay. Nevertheless, taken together, the results of these three independent analyses revealed that mTEAD-2 was selectively expressed during preimplantation development.

Only mTEAD-2 RNA was detected in embryos up to day 7

To determine whether other members of the mTEAD gene family were expressed later in development, mTEAD-1, 2, and 3 RNAs were assayed by in situ hybridization in day-7 embryos, which were sectioned within the decidua. In situ hybridization of serial sections revealed that both mTEAD-1 and mTEAD-3 were expressed only in the decidual cells; not in the embryo (Fig. 6D-I). Antisense probe for mTEAD-3 stained the anti-mesometrial portion of the deciduum, but not the embryo (Fig. 6G,I). mTEAD-1 mRNA was detected throughout the deciduum, but not in the embryo (Fig. 6D,F). None of the three sense mTEAD probes stained any portion of either deciduum or embryo (Fig. 6B,E,H). In contrast, mTEAD-2 was expressed in the embryo, as well as in the mesometrial portion of the deciduum (Fig. 6A-C). The mTEAD-2 antisense probe stained cells uniformly throughout the embryo (Fig. 7), while the mTEAD-2 sense probe failed to stain any of the sections (data not shown). Interestingly, staining appears to be reduced in the extraembryonic regions (Fig. 6C). Thus, only mTEAD-2 RNA was detected in the embryo at day 7 of gestation.

Fig. 6.

Distribution of mTEAD mRNAs in mouse decidua containing a day-7 embryo. Digoxigenin-labeled RNA probes specific for mTEAD-2 (A-C), mTEAD-1 (D-F), or mTEAD-3 (G-I) were synthesized from the C-terminal half of the respective cDNAs, thereby avoiding regions containing the conserved TEA DNA binding domain. In situ hybridization of a deciduum containing day-7 embryos using adjacent frozen serial sections through the entire deciduum (devoid of the myometrium) was carried out with antisense (A,D,G) or sense (B,E,H) probes. Higher magnifications (6.4×) of the embryonic regions from A,D and G are shown in C,F and I, respectively. Cells representing the mesometrial deciduum (MM), antimesometrial deciduum (AMM) and embryo are indicated in A. Bar represents 50 μm.

Fig. 6.

Distribution of mTEAD mRNAs in mouse decidua containing a day-7 embryo. Digoxigenin-labeled RNA probes specific for mTEAD-2 (A-C), mTEAD-1 (D-F), or mTEAD-3 (G-I) were synthesized from the C-terminal half of the respective cDNAs, thereby avoiding regions containing the conserved TEA DNA binding domain. In situ hybridization of a deciduum containing day-7 embryos using adjacent frozen serial sections through the entire deciduum (devoid of the myometrium) was carried out with antisense (A,D,G) or sense (B,E,H) probes. Higher magnifications (6.4×) of the embryonic regions from A,D and G are shown in C,F and I, respectively. Cells representing the mesometrial deciduum (MM), antimesometrial deciduum (AMM) and embryo are indicated in A. Bar represents 50 μm.

Fig. 7.

Distribution of mTEAD-2 mRNA within a single day-7 mouse embryo. In situ hybridization was carried out as described in Fig. 6, except that adjacent serial sections through a single deciduum containing a day-7 embryo were hybridized with antisense mTEAD-2 probes. Sense probe showed no specific staining (data not shown). Bar represents 50 μm.

Fig. 7.

Distribution of mTEAD-2 mRNA within a single day-7 mouse embryo. In situ hybridization was carried out as described in Fig. 6, except that adjacent serial sections through a single deciduum containing a day-7 embryo were hybridized with antisense mTEAD-2 probes. Sense probe showed no specific staining (data not shown). Bar represents 50 μm.

Because previous studies showed that mTEAD-1 gene function is required by around day 10 (Chen et al., 1994), northern blotting hybridization was used to examine mTEAD gene expression at day 9 and 10 of development (Fig. 8). These results revealed that although mTEAD-2 RNA was expressed more abundantly, mTEAD-1 RNA also was expressed at least by day 9. mTEAD-3 RNA also appeared to be expressed at low but detectable levels. Thus, mTEAD-1 RNA appeared concurrent with the requirement for its function during mouse development.

Fig. 8.

Detection of mTEAD RNAs in mouse embryos and adult tissues. Total RNA was prepared from day-9 and day-10 mouse embryos that had been dissected away from their surrounding decidua (Hogan et al., 1994). The RNA was then fractionated by denaturing gel electrophoresis, transferred to a membrane, and hybridized first with a 32P-labeled DNA probe specific for mTEAD-2 This blot was stripped and hybridized with a probe specific for mTEAD-1, then stripped and hybridized with a probe specific for mTEAD-3, and finally stripped and hybridized with a probe specific for β-actin. The sizes of RNA standards that were fractionated in parallel are indicated in kilobases.

Fig. 8.

Detection of mTEAD RNAs in mouse embryos and adult tissues. Total RNA was prepared from day-9 and day-10 mouse embryos that had been dissected away from their surrounding decidua (Hogan et al., 1994). The RNA was then fractionated by denaturing gel electrophoresis, transferred to a membrane, and hybridized first with a 32P-labeled DNA probe specific for mTEAD-2 This blot was stripped and hybridized with a probe specific for mTEAD-1, then stripped and hybridized with a probe specific for mTEAD-3, and finally stripped and hybridized with a probe specific for β-actin. The sizes of RNA standards that were fractionated in parallel are indicated in kilobases.

mTEAD-2 was also expressed in some adult tissues

The above results raised the possibility that mTEAD-2 expression is confined to early embryos, while other members of this transcription factor gene family are expressed in later embryos and adult tissues. Therefore, RNA expression patterns of mTEAD genes were examined in adult mouse tissues using northern blotting hybridization analysis of poly(A)+ RNA (Fig. 8). Each mTEAD mRNA was identified by hybridization to its cognate probe under conditions in which only the indicated gene mRNA was recognized. In contrast to mTEAD-4 (TEFR-1), whose expression was reported to be restricted to certain tissues (Yockey et al., 1996), mTEAD-1, 2, and 3 genes were ubiquitously expressed in adult tissues, with marked differences existing among their relative levels of expression.

In agreement with previously published results (Blatt and DePamphilis, 1993; Shimizu et al., 1993), mTEAD-1 mRNA was expressed in most mouse tissues, ranging from very high levels in lung, muscle, kidney and heart to low levels in brain, spleen, liver and testes (Fig. 8). However, while all three mTEAD genes were expressed at high levels in lung, and at lower levels in heart and spleen, mTEAD-1 and 3 were expressed strongly in kidney and liver, whereas mTEAD-1 and 2 were preferentially expressed in brain. Furthermore, these and other results show that mTEAD-2 was not confined to embryonic tissues, since it was strongly expressed in testes (Fig. 8) and ovarian follicle cells (Fig. 2). A previous report that mTEAD-2 (ETF) is expressed only in embryonic tissues (Yasunami et al., 1995) may have resulted from their analysis of total RNA instead of poly(A)+ mRNA. Thus, while at least one member of the mTEAD gene family was expressed in most, perhaps all, adult mouse tissues, distinct patterns of expression existed for individual family members, suggesting that each of the four genes had a distinct function.

TEAD transcription factor activity first appears at the onset of zygotic gene expression

The most critical event to occur following fertilization is the activation and precise timing of zygotic transcription. This event is essential for viability of the embryo, and its timing must be precisely controlled. Recent studies indicate that the stage-specific production of key transcription factors likely plays a central role in controlling and mediating this genome activation (Wang and Latham, 1997). The experiments presented here identify one of the first transcription factors (mTEAD-2) expressed specifically when zygotic genes are activated in mouse embryos. Enhancers whose activity depends on the presence of one or more mTEAD DNA binding sites (GTIIC sequence; Davidson et al., 1988) are the most powerful transcription enhancers so far identified when plasmid encoded genes are injected into 2-cell mouse embryos (Martinez-Salas et al., 1989; Melin et al., 1993). Similar studies in the absence of aphidicolin have shown that mTEAD-dependent enhancer activity is present at least up to the morula stage in development, and transfection studies have identified this activity in ES cells, suggesting that it is present in the inner cell mass of blastocysts (Martinez-Salas et al., 1989; Melin et al., 1993). Therefore, it is not surprising that a promoter constructed from a tandem series of mTEAD binding sites was as active in cleavage stage mouse embryos as promoters that depended on Sp1 (Fig. 1), a ubiquitous transcription factor active in mouse oocytes and early embryos (Majumder et al., 1993; Worrad et al., 1994). The level of luciferase activity in these experiments depends on the number and composition of the transcription factor binding sites present, and on the presence of transcription factors that bind to these sites (Majumder et al., 1993, 1997). The TATA box element does not contribute to the level of promoter activity in cleavage stage embryos (Majumder and DePamphilis, 1994), although a TATA box alone is about 10% as active as a complete promoter (data not shown; Majumder et al., 1993).

What is surprising is that mTEAD activity was reduced in arrested 1-cell embryos and undetectable in oocytes; both of which yield easily detected levels of Sp1-dependent promoter activity (Fig. 1). The level of luciferase produced from paternal pronuclei in S-phase arrested 1-cell embryos reflects the level of promoter activity at the onset of zygotic gene expression at the 2-cell stage in the absence of chromatin mediated repression (Majumder and DePamphilis, 1995; Nothias et al., 1995). Therefore, we conclude that the appearance of TEAD transcription factor occurs concomitant with the onset of zygotic gene expression.

mTEAD-2 appears to be the only member of the TEAD transcription factor family expressed in early mouse embryos

Results presented here support the conclusion that mTEAD-dependent transcription factor activity in preimplantation embryos most likely results from zygotic expression of mTEAD-2 genes. mTEAD transcription factor activity was not detected in oocytes, suggesting that maternally inherited transcription factors do not contribute to mTEAD activity in early cleavage stage embryos. mTEAD-4 RNA was not detected in either oocytes or preimplantation embryos, and the low levels of maternal mTEAD-1 and 3 RNA that were detected in oocytes were essentially eliminated by the 2-cell stage (Figs 3, 4, 5), consistent with previous studies on degradation of mouse maternal mRNAs (Ebert et al., 1984). mTEAD transcription factor activity was detected at low levels relative to constitutive transcription factor Sp1 in arrested 1-cell embryos and to a much higher level in 2-cell/4-cell embryos (Fig. 1D), consistent with expression of a zygotic gene. While it is possible that transient expression of maternally inherited mTEAD-1 or 3 RNA contributes to the initial mTEAD transcription factor activity seen in early cleavage embryos, it seems more likely that this activity results from expression of zygotic mTEAD-2 genes. Only mTEAD-2 mRNA accumulated during subsequent preimplantation development, reaching levels at least 100-fold greater than those for mTEAD-3 RNA. Moreover, mTEAD-2 was the only mTEAD gene expressed at detectable levels in day-7 embryos. The sharp rise in mTEAD-2 mRNA that was observed with 8-cell rather than 2-cell embryos can be explained as follows. First, maternal mTEAD-2 RNA was likely being degraded while zygotic RNA was being produced. Second, assuming that TEAD-2 is expressed uniformly throughout preimplantation embryos (as it is in embryos at day 7; Fig. 7), the amount of mTEAD-2 RNA per embryo will increase exponentially as cell cleavage occurs. Thus, the early increase in mTEAD activity at the 2-cell stage and later, observed with injected reporter genes, can be accounted for by the temporal pattern of mTEAD-2 expression.

Nevertheless, the initial phase of mTEAD transcription factor activity seen in early cleavage embryos could come from maternal RNA or proteins. The apparent absence of mTEAD activity in oocytes was not due to an inability of oocytes to utilize injected plasmid encoded promoters, because activity from Sp1-dependent promoters was easily detected. Neither was it due to insufficient levels of oocyte mTEAD-2 mRNA, because the levels of mTEAD-2 mRNA in oocytes and 1-cell embryos were at least twice the levels in 2/4-cell embryos (Fig. 4C). Therefore, mTEAD mRNAs detected in oocytes may not participate until later in development. Since one mechanism of regulating ZGE may involve the delayed translation of maternal mRNAs that encode transcription factors (Wang and Latham, 1997), it is possible that mTEAD-1, 2 or 3 maternal RNA might be translated prior to or concomitant with ZGE. Alternatively, translated mTEAD transcription factors may not become active until a specific cofactor is expressed at ZGA (Majumder et al., 1997).

The levels of mTEAD-2 mRNA in oocytes and early cleavage embryos [2000 to 6000 copies per ovum or embryo (Fig. 4C)] were substantially lower than the levels observed at later times during development. In fact, in situ hybridization analysis failed to detect any of the three mTEAD mRNAs in oocytes, although mTEAD-2 was readily apparent in the granulosa cells surrounding the oocytes (Fig. 2). Therefore, approx. 5000 copies of mRNA per cell was below the level of detection for our in situ hybridization protocol. Using in situ hybridization analysis, only mTEAD-2 mRNA was detected in day-7 embryos (Fig. 6) and it was distributed throughout the embryo (Fig. 7), suggesting that mTEAD-2 was the only member of this gene family required for early embryonic development. While these data also suggested that mTEAD-2 was not expressed in extraembryonic tissues, the frozen sections used in this study lacked sufficient morphological detail to allow definitive conclusions on mTEAD-2 expression in the individual tissues of the gastrulating embryo.

Jacquemin et al. (1996) also found that mTEAD-2 RNA was expressed abundantly throughout a day 6.5 d.p.c. conceptus (equivalent to our day-7 decidua), and that mTEAD-2 expression in the extraembryonic tissues declined between 6.5 and 8.5 d.p.c., consistent with the observations reported here. However, they also reported that mTEAD-1 was expressed at 6.5 d.p.c., but this expression was barely detectable and highly localized, while mTEAD-2 expression was strong and uniformly distributed throughout their embryos. While it is possible that the TEAD-1 promoter is active prior to day 9, as suggested by the expression of a reporter gene downstream of the mTEAD-1 promoter (Chen et al., 1994), both RT-PCR (Figs 3, 4) and in situ hybridization (Figs 6, 7) analyses show that the level of mTEAD-1 promoter activity is quite low relative to that of the mTEAD-2 gene up to day 7 of development. Therefore, we conclude that mTEAD-2 is expressed pre-dominantly, if not exclusively, in developing embryos up to day 7, while mTEAD-1 (Fig. 8; Jacquemin et al., 1996), mTEAD-3 (Fig. 8; Yasunami et al., 1996) and mTEAD-4 (Jacquemin et al., 1996; Yasunami et al., 1996; Yockey et al., 1996) expression begins sometime from day 7 to day 9.

Taken together, these observations provide an explanation for why mTEAD-1 is not required during mouse development until about day 10 (Chen et al., 1994), even though DNA binding sites for this gene product can efficiently stimulate transcription in preimplantation embryos. Moreover, the fact that mTEAD-1 function around day 10 cannot be carried out by mTEAD-2, argues that these two genes are not functionally redundant. Such non-redundant roles by members of this gene family is also supported by their tissue/cell-specific expression seen in adults (Figs 2, 6, 8; Yockey et al., 1996).

Identification and extensive analyses of mouse transcription factors expressed during preimplantation development are pre-requisites for understanding, at the molecular level, these early stages of development. To date, relatively few of such transcription factors have been identified. Mash-2 expression, while detectable during the preimplantation stage, becomes restricted to the trophectoderm in blastocysts and is not required for preimplantation development (Guillemot et al., 1994). Only one other transcription factor has been reported with expression characteristics similar to that of mTEAD-2. Oct-4 is a transcription factor present in both oocytes and preimplantation embryos and is down-regulated in trophoblasts of the late blstocysts (Palmieri et al., 1994). Changes in Oct-4 mRNA are similar to those described here for mTEAD-2. Both mRNAs are present in oocytes, decrease during the 2-cell stage and then dramatically increase between the 4-cell and 8-cell embryonic stage (Fig. 4; Scholer, 1991). The fact that microinjection studies detected mTEAD-dependent transcription factor activity at the beginning of the 2-cell stage demonstrated that significant mTEAD-2 expression occurred upon zygotic gene activation. Whether or not the same is true for Oct-4 remains to be determined. Mice also encode an activity that is similar to adenovirus E1A protein. This activity is restricted to oocytes and preimplantation embryos (Dooley et al., 1989), and may interact with Oct-4 (Scholer et al., 1991). Thus, mTEAD-2 is one of the first transcription factors to be expressed specifically at the onset of zygotic gene expression, and only the second such activity identified in mammalian development. Presumably, the purpose of mTEAD-2 and Oct-4 transcription factors is to activate genes that are required for further preimplantation development.

We would like to acknowledge M. Miranda for her technical expertise in microinjection experiments, and C. Stewart and the members of DePamphilis laboratory for helpful discussions. We would also like to thank Dr. N. Shimizu for sending us the cDNA for TEFR1a. This work was supported in part (awarded to K. E. L.) by International Foundation for Ethical Research, National Science Foundation (MCB-9630370).

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