The Japanese Society of Developmental Biologists
Develop. Growth Differ. (2013) 55, 776–785
doi: 10.1111/dgd.12100
Original Article
Transcriptional regulation in the early ectodermal lineage of ascidian embryos Yosuke Horikawa, Haruka Matsumoto, Fumika Yamaguchi, † Satomi Ishida and Shigeki Fujiwara* Department of Applied Science, Kochi University, 2-5-1 Akebono-cho, Kochi-shi, Kochi, 780-8520, Japan
In ascidian embryos, ectodermal tissues derive from blastomeres in the animal hemisphere. The animal hemisphere-specific gene expression is observed as early as the 16-cell stage. Here, we characterized animal hemisphere-specific enhancers of three genes, Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4. Deletion analyses identified minimal essential elements. Although these elements contained multiple GATA sequences, electrophoretic mobility shift assays revealed that only some of them were strong binding sites for the transcription factor Ci-GATAa. On the other hand, the motif-searching software MEME identified an octamer, GA (T/G) AAGGG, shared by these enhancers. In Ci-ephrin-Ad and Ci-TGFb-NA1, the octamer was GATAAGGG, which strongly bound Ci-GATAa. The 397-bp upstream region of Ci-ephrin-Ad contained two strong Ci-GATAa-binding sites, one of which was the octamer motif. Mutation in the octamer motif, but not the other Ci-GATAa-binding site, severely affected the enhancer activity. The 204-bp upstream region of Ci-TGFb-NA1 contained four strong Ci-GATAa-binding sites, including the octamer motif. Mutation only in the octamer motif, leaving the other three Ci-GATAa-binding sites intact, abolished the enhancer activity. These results suggest a crucial role for the octamer motif. Key words: ascidian, ectoderm, enhancer, GATA, transcriptional activation.
Introduction The ectoderm arises from the animal hemisphere in chordates, including amphibians and ascidians (Lemaire et al. 2008). It is subsequently subdivided into the epidermis and central nervous system. The mechanism of neural induction has been extensively studied (for reviews see Stern 2005; Schmidt et al. 2013). Factors promoting epidermal specification and suppressing neural fate have also been identified, which are activated by bone morphogenetic protein (e.g. Qiao et al. 2012; Trıbulo et al. 2012). However, few genes have been identified that were expressed in the uncommitted general ectoderm. Our knowledge about *Author to whom all correspondence should be addressed. Email: [email protected] † Present address: Laboratory of Molecular Biology, Medical Research Center, Kochi Medical School, Kochi, 783-8505, Japan. Received 3 September 2012; revised 9 October 2013; accepted 10 October 2013. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists
the mechanisms of specification of the epidermis and general ectoderm is fragmentary. The third cleavage of ascidian embryos divides the embryo into four animal and four vegetal blastomeres. The animal blastomeres mainly give rise to the epidermis and central nervous system (Conklin 1905; Nishida 1987). Nishikata et al. (2001) reported that mRNAs of a few genes were predominantly localized in the animal blastomeres of the 8-cell embryo, although it is not clear whether this localization is achieved by specific zygotic expression or asymmetrical distribution of maternal mRNA. The mRNAs localized to the animal blastomeres include those encoding a WD repeat-containing protein, an F-actin-capping protein, a calcineurin inhibitor, and a putative GTP-binding protein (Nishikata et al. 2001). It is unclear whether these proteins are involved in the ectodermal genetic regulatory cascade. At the 16-cell stage, many genes are expressed exclusively in the animal hemisphere. These include the genes encoding transcription factors; Ci-SoxF €cher et al. 2007). (Imai et al. 2004) and Ci-fog (Rothba Components of cell–cell signaling pathways are also expressed in the animal hemisphere; Ci-ephrin-Aa, Ci-ephrin-Ad, Ci-TGFb-NA1, Ci-Smad1/5 and Ci-Fz4
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(Imai et al. 2004; Hamaguchi et al. 2007; Picco et al. 2007). Cell adhesion molecules (Ci-d1-protocadherinlike and Ci-d-protocadherin-4) are also specifically expressed (Noda & Satoh 2008). Transcription of Ci-fog in the animal hemisphere is activated by the transcription factor Ci-GATAa €cher et al. 2007). A Ci-GATAa-specific mor(Rothba pholino oligo suppresses the enhancer activity of €cher et al. 2007). Transcriptional activaCi-fog (Rothba tion of Ci-otx in the animal hemisphere also requires Ci-GATAa (Bertrand et al. 2003). The enhancer regions of these genes contain multiple GATA sequences (Ber€cher et al. 2007), although trand et al. 2003; Rothba binding of the Ci-GATAa protein to these sequences was not demonstrated. In addition, it is not yet clear whether only Ci-GATAa is responsible for activation of animal hemisphere-specific genes. In the present study, we identified animal hemispherespecific enhancer elements within the 5′ flanking regions of three genes (Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4). These genes were chosen because their specific expression was clear and strong (Imai et al. 2004; Picco et al. 2007). We found multiple GATA sequences in the upstream regions of these genes. Electrophoretic mobility shift assays revealed that some, but not all, of them strongly bind the Ci-GATAa protein. However, disruption of only one of them, whose GATA sequence was followed by AGGG, severely affected the enhancer activity. Based on these results, the possible involvement of transcription factor (s) other than Ci-GATAa is discussed.
Materials and methods Animals Juvenile adults of Ciona intestinalis were purchased from the National Bio-Resource Project of MEXT, Japan. The animals were cultured in Tosa Bay near the Usa Marine Biological Institute of Kochi University. Eggs and sperm were obtained from the gonoducts of mature adults. Eggs were inseminated with non-self sperm. Fertilized eggs were dechorionated with 0.05% actinase E (Kaken Pharmaceutical) and 1% sodium thioglycolate. Embryos were reared in artificial seawater Super Marine Art SF1 (Tomita Pharmaceutical). Construction of plasmids Genomic DNA fragments corresponding to the promoter region of Ci-ephrin-Ad, Ci-TGFb-NA1 and CiFz4 were amplified by polymerase chain reaction (PCR), using the primers ephrin1.7F and ephrin1.7R for Ci-ephrin-Ad, tgfna3.0F and tgfna3.0R for
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Ci-TGFb-NA1, and fz2.0F and fz2.0R for Ci-Fz4 (Table S1). The PCR products were inserted into pGEM-T (Promega). The Ci-ephrin-Ad fragment was excised with PstI and BamHI from pGEM-T, while the Ci-TGFb-NA1 and Ci-Fz4 fragments were excised with XhoI and BamHI. These fragments were inserted into pSP72-1.27 (Corbo et al. 1997). The resultant plasmids were named Ephrin1648Z, Tgfna2940Z and Fz1964Z, respectively. For construction of Ephrin545Z, Ephrin497Z, Ephrin447Z, Ephrin397Z and Ephrin333Z, PCR was carried out using a lacZ-specific primer (gal-A2) and a Ci-ephrin-Ad-specific primer (ephrin545F, ephrin497F, ephrin447F, ephrin397F and ephrin333F, respectively; Table S1). Ephrin1648Z was used as a template. For construction of Tgfna799Z, Tgfna599Z, Tgfna399Z, Tgfna204Z and Tgfna66Z, PCR was carried out using tgfna3.0R and one of the following primers (tgfna799F, tgfna599F, tgfna399F, tgfna204F and tgfna66F, respectively; Table S1). Tgfna2940Z was used as a template. For construction of Fz1425Z, Fz1294Z, Fz975Z, Fz784Z and Fz387Z, PCR was carried out using fz2.0R and one of the following primers (fz1425F, fz1294F, fz975F, fz784F and fz387F, respectively; Table S1). Fz1964Z was used as a template. PCR products were once inserted into pGEM-T, excised with XhoI and BamHI, and inserted into pSP72-1.27. Site-directed mutagenesis was performed according to the protocol described by Fujiwara et al. (1998). To create point mutations within the Ci-ephrin-Ad enhancer, the XhoI-BamHI fragment of Ephrin397Z was inserted into pBluescript II SK+ (Stratagene). Uracilcontaining single-stranded DNA was prepared using the Escherichia coli strain CJ236 (Takara). Mutagenic oligonucleotides used were E-S1 m, E-S2 m, E-G1 m, E-G2 m, E-S1G1 m and E-Mm (Table S1). For point mutations in the Ci-TGFb-NA1 enhancer, we transformed CJ236 with pGEM-T containing the PCR product using primers tgfna204F and tgfna3.0R (described above). Mutagenic oligonucleotides used were T-Gm and T-Mm (Table S1). Electroporation and detection of transgene expression Plasmid DNA was prepared using QIAGEN tip-100 (Qiagen). Transgenes were introduced into dechorionated embryos by electroporation, according to the protocol described by Corbo et al. (1997). Expression of lacZ was detected by in situ hybridization as described by Kanda et al. (2009). In each experiment, 100–400 electroporated embryos were observed under an AZ100 microscope (Nikon). Embryos expressing lacZ in one or more blastomeres were counted as positive.
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The percentage of positive embryos among morphologically normal embryos was calculated. Electrophoretic mobility shift assay The entire translated region of Ci-GATAa was amplified by PCR using the cDNA clone “cien229100” as a template (Tassy et al. 2010; http://www.aniseed.cnrs.fr/ index.php). The PCR was carried out using primers GATA-F and GATA-Nhe-R (Table S1), and Tks Gflex DNA polymerase (Takara), according to the protocol supplied by the manufacturer. The product of the PCR was briefly treated with Taq DNA polymerase (Ampliqon) to create the 3′ overhang of a single adenine nucleotide, and inserted into pGEM-T. The Ci-GATAa cDNA was again amplified using Tks Gflex DNA polymerase and the primers GATA-F and M13-Forward-40 (5′-GTTTTCCCAGTCACGAC-3′). The product of the PCR was excised with NheI. The pCMX-hRARa plasmid (Umesono et al. 1991) was digested with MscI and NheI to remove the cDNA encoding human retinoic acid receptor a (hRARa). The Ci-GATAa cDNA was inserted into the pCMX vector. The Ci-GATAa protein was produced using the TNT coupled reticulocyte lysate system (Promega). Double-stranded DNA
probes were labeled with [c-32P] adenosine triphosphate (Perkin Elmer Japan), according to the protocol described by Kanda et al. (2009). Electrophoretic mobility shift assays (EMSAs) were carried out using a Gel-shift assay system (Promega). Autoradiograms were obtained using BAS2500 (Fuji Film).
Results Identification of early animal-hemisphere enhancers A fragment of the C. intestinalis genomic DNA was obtained, which contained the promoter region of Ciephrin-Ad, including putative transcription and translation initiation sites (Fig. 1a). The fragment was fused in-frame with lacZ and introduced into fertilized eggs by electroporation (Fig. 1b). The resultant transgene, named Ephrin1648Z, contained 1648 bp of the 5′ flanking region, according to the gene model KH.C3.716.v1.A.SL1-1 in the C. intestinalis genome database (Satou et al. 2005; http://ghost.zool.kyoto-u. ac.jp/SearchGenomekh.html). Ephrin1648Z was introduced into one-cell embryos. The embryos were fixed at the 16-cell stage and the lacZ mRNA was detected by in situ hybridization. Ephrin1648Z was expressed in
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Fig. 1. The animal hemisphere enhancer of Ci-ephrin-Ad. (a) The genomic structure of the Ci-ephrin-Ad gene, predicted in the Ciona intestinalis genome database (Satou et al. 2005; http://ghost.zool.kyoto-u.ac.jp/SearchGenomekh.html). Exons are indicated by boxes. The open reading frame is in green. The 1.6-kb upstream region examined in the present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The E1 enhancer is indicated by a red box. Percentage of embryos expressing lacZ among normally developing embryos is indicated. (c–h) The expression pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up. Names of the blastomeres are indicated in (c). ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
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all of the blastomeres (a5.3, a5.4, b5.3 and b5.4 blastomere pairs) in the animal hemisphere (Fig. 1c). Transgenes containing 545, 497, 447, or 397 bp of the upstream sequence gave essentially similar patterns of expression, although some embryos did not express lacZ in all of the eight blastomeres (Fig. 1d–g). No embryo carrying Ephrin333Z expressed lacZ in any blastomere (Fig. 1h). These results indicate that 397 bp of the 5′ flanking region of Ci-ephrin-Ad is sufficient, and the region between 397 and 334, named E1, is necessary for transcriptional activation in the animal hemisphere. We also isolated the promoter regions of Ci-TGFb-NA1 and Ci-Fz4, and placed them upstream of lacZ. (Figs 2 and 3). A transgene named Tgfna2940Z contained 2940 bp of the 5′ flanking region of Ci-TGFb-NA1, according to the gene model KH.C4.547.v1.A.nonSL1-1 (Fig. 2a,b). Tgfna2940Z was expressed in all of the eight blastomeres in the animal hemisphere of the 16-cell embryo (Fig. 2c). Deletion analyses revealed that 204 bp of the upstream sequence of Ci-TGFbNA1 was sufficient, and the region between 204 and 66, named T1, was necessary for transcriptional activation in the animal hemisphere (Fig. 2d–h). Fz1964Z contained 1964 bp of the 5′ flanking region of Ci-Fz4, according to the gene model KH.C6.162.v2.A. SL2-1 (Fig. 3a,b). Fz1964Z recapitulated the animal (a)
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hemisphere-specific expression of Ci-Fz4 (Fig. 3c). Deletion analyses revealed that 975 bp of the upstream sequence of Ci-Fz4 was sufficient, and the region between 975 and 784, named F1, was necessary for transcriptional activation in the animal hemisphere (Fig. 3d–h). The region between 783 and 67 was deleted from Fz975Z. The resultant transgene was not expressed, suggesting that the proximal region (downstream to 784) also contains enhancer element (s) necessary for transcriptional activation (data not shown). Possible binding sites for transcription factors We searched for sequence motifs shared by Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4, using MEME (Bailey & Elkan 1994; http://meme.sdsc.edu/meme/intro.html). MEME identified an octamer sequence, GA (T/G) AAGGG. The E1 and T1 enhancers contained a single octamer motif each (GATAAGGG), named E-M1 and T-M1, respectively (Fig. 4). MEME did not find this motif within the 975-bp proximal region of Ci-Fz4. However, we found a similar sequence, GAAAAGGG, at nucleotide position 73 of Ci-Fz4 (data not shown). We also searched for putative binding sites for known transcription factors. Ci-GATAa is necessary for activation of genes in the animal hemisphere of the Ciona (c)
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Fig. 2. The animal hemisphere enhancer of Ci-TGFb-NA1. (a) The genomic structure of the Ci-TGFb-NA1 gene, predicted in the Ciona intestinalis genome database. Exons are indicated by boxes. The open reading frame is in green. The 2.94-kb upstream region examined in the present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The T1 enhancer is indicated by a red box. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (c–h) The expression pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up. ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
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Fig. 3. The animal hemisphere enhancer of Ci-Fz4. (a) The genomic structure of the Ci-Fz4 gene, predicted in the Ciona intestinalis genome database. Exons are indicated by boxes. The open reading frame is in green. The 1.96-kb upstream region examined in the present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The F1 enhancer is indicated by a red box. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (c–h) The expression pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up.
€cher et al. 2007). The binding embryo (Rothba sequence for Ci-GATAa is 5′-GATA-3′, and it had no strong constraint on surrounding sequences (K. Nitta & P. Lemaire, pers. comm., 2012). Ci-SoxF is the only transcription factor whose mRNA is exclusively detected in the animal hemisphere at the 16-cell stage (Imai et al. 2004). The core recognition sequence for the Sox group transcription factors is AACAAT (Mertin et al. 1999). In vitro binding experiments revealed that the consensus sequence for C. intestinalis Sox proteins is (A/G) (A/C) CAA (T/A) (K. Nitta & P. Lemaire, pers. comm., 2012). We found six GATA-binding sites and two Sox-binding sites within the 397-bp proximal region of Ci-ephrin-Ad (Fig. 4a). The E1 enhancer contained two GATA-binding sites (named E-G1 and EG2) and one Sox-binding site (named E-S1; Fig. 4a,b). The 204-bp upstream region of Ci-TGFb-NA1 contained no Sox-binding site, but four GATA-binding sites (Fig. 4a). The T1 enhancer contained three of them (named T-G1–T-G3; Fig. 4c). E-M1 and T-M1 included the GATA sequence (E-G2 and T-G3, respectively, Fig. 4). The octamer motif is necessary for enhancer activity Since the E1 enhancer is necessary for transcriptional activation in the animal hemisphere, we tested the
requirement of putative binding sites for transcription factors by site-directed mutagenesis. First, a point mutation was generated at the Sox-binding site within the E1 enhancer (E-S1; Fig. 5a,b). The mutant transgene, named Ephrin (S1 m) Z, was expressed normally in the animal hemisphere at the 16-cell stage (Fig. 5b). Another possible Sox-binding sequence was found downstream of nucleotide position 333 (E-S2; Fig. 5a). Disruption of E-S2, or a double mutation in E-S1 and E-S2, did not affect the expression in the animal hemisphere (Fig. 5c,d). One of the two GATAbinding sites within the E1 enhancer (E-G1) was mutagenized (Fig. 5a,e). The mutant transgene, named Ephrin (G1 m) Z, was expressed in the animal hemisphere (Fig. 5e). In contrast, a mutation in the other GATA sequence (E-G2) caused complete silencing of the reporter gene (Fig. 5f). Note that E-G2 overlaps with E-M1 (GATAAGGG) (Figs. 4b and 5a), and that a point mutation in E-G2 completely inactivated the enhancer that still contained five additional GATA sequences (see Fig. 4a). We then introduced a point mutation into E-M1 without disrupting E-G2 (GATAAGGG to GATActtt). The resultant transgene, named Ephrin (M1 m) Z, was not expressed in any blastomere (Fig. 5g). About 83% of the embryos carrying Ephrin397Z expressed lacZ, while only 14% of the embryos carrying Ephrin (M1 m) Z expressed it.
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DNA/protein complex (Fig. 7a, lane 2). We prepared a mutant probe (E-M1 m), in which AGGG of E-M1 was disrupted without affecting GATA. E-M1m did not form a complex with Ci-GATAa (Fig. 7a, lane 3). A probe containing E-G1 formed a complex with Ci-GATAa (Fig. 7a, lane 4). E-G3, E-G5 and E-G6 formed a weak band (Fig. 7a, lanes 5, 7, 8). No specific shift was observed with E-G4 (Fig. 7a, lanes 6). A probe containing T-M1 (overlapping with T-G3) and T-G2 formed a complex with Ci-GATAa (Fig. 7b, lane 2). The shifted band became weak when mutation was created in either T-G2 or T-G3 (Fig. 7b, lanes 3, 5). When the AGGG moiety of T-M1 was disrupted without affecting GATA, the complex’s formation was also affected (Fig. 7b, lane 4). No shifted band was observed when both T-G2 and T-G3 were mutated (Fig. 7b, lane 6). T-G1 and T-G4 also strongly formed a complex with Ci-GATAa (Fig. 7b, lanes 7, 8).
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Discussion Binding of Ci-GATAa is strongly influenced by flanking sequences
Fig. 4. Putative transcription factor-binding sites in the E1 and T1 enhancers (a) Schematic diagrams showing the 5´ flanking regions of Ci-ephrin-Ad and Ci-TGFb-NA1. Putative binding sites for the GATA and Sox transcription factors are indicated by blue and yellow boxes, respectively. The octamer motif (GATAAGGG) is indicated by red boxes. (b) Nucleotide sequence of the E1 enhancer. (c) Nucleotide sequence of the T1 enhancer.
The T1 enhancer contains three GATA sequences, one of which (T-G3) overlaps with T-M1 (GATAAGGG) (Figs 4c and 6a). Disruption of T-G3 (GATAAGGG to GtaAAGGG) completely silenced the expression of lacZ (Fig. 6b). A point mutation of T-M1 that did not disrupt T-G3 (GATAAGGG to GATActtt) also caused severe inactivation of the enhancer (Fig. 6c). About 81% of the embryos carrying Tgfna204Z expressed lacZ, while 3.6% of the embryos carrying Tgfna (M1 m) Z did so. Binding of the Ci-GATAa protein to wild-type and mutagenized DNA sequences Electrophoretic mobility shift assays were carried out using a full-length Ci-GAGAa protein synthesized in vitro. An oligonucleotide probe containing the E-M1 (GATAAGGG) and its flanking sequences formed a
Direct binding assays of the ascidian GATA protein were first reported in the present study. Our EMSA revealed cases in which the affinity of the Ci-GATAa binding to DNA was strongly affected by the sequences surrounding the core GATA sequence. The 397-bp upstream region of Ci-ephrin-Ad contains six GATA sequences. However, only two of them (E-G1 and E-G2) can strongly bind Ci-GATAa. These results seem inconsistent with unpublished results obtained by K. Nitta and P. Lemaire (pers. comm., 2012). They searched for a consensus Ci-GATAa-binding sequence by the systematic evolution of ligands using exponential enrichment (SELEX). They did not see a strong preference outside of the GATA sequence, although TGATAA was a little better (K. Nitta & P. Lemaire, pers. comm., 2012). In the present study, Ci-GATAa seemed to prefer GATAAGGG. The underlined nucleotides may not have been recognized by SELEX, possibly because only two or three of the underlined nucleotides are conserved in many cases. €cher et al. (2007) Bertrand et al. (2003) and Rothba carried out mutagenesis of many GATA sequences within the enhancer region of Ci-otx and Ci-fog, and reported strong suppression of reporter genes. Similar results were obtained by antisense-mediated knock€cher down of Ci-GATAa (Bertrand et al. 2003; Rothba et al. 2007). Two critical GATA sequences within the Ci-fog enhancer are GATAAAGT and GATACTTT €cher et al. 2007). The former is similar to the (Rothba octamer motif, but the latter looks like the mutant
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(g) Fig. 5. Effect of point mutations in putative transcription factor-binding sites in the 5´ flanking region of Ci-ephrin-Ad. Diagrams of transgenes and their pattern of expression are shown side-by-side. Percentage of the embryos expressing lacZ among normally developing embryos is also indicated. (a) Ephrin397Z, carrying no mutation. (b) Ephrin (S1 m) Z, carrying a mutation in the Sox-binding site ES1. (c) Ephrin (S2 m) Z, carrying a mutation in the other Sox-binding site E-S2. (d) Ephrin (S1S2 m) Z, carrying mutations in both E-S1 and E-S2. (e) Ephrin (G1 m) Z, carrying a mutation in the GATA sequence E-G1. (f) Ephrin (G2 m) Z, carrying a mutation in the GATA sequence E-G2, which overlaps with the octamer motif E-M1. (g) Ephrin (M1 m) Z, carrying a mutation in E-M1. The mutation was created to convert GATAAGGG into GATATCCC, not to disrupt E-G2.
sequences that did not bind Ci-GATAa in the present study. None of the seven GATA sequences within the Ci-otx enhancer look similar to the strong binding sites (Bertrand et al. 2003). Considering the present results, direct binding assays will be necessary to confirm that these GATA sequences are genuine Ci-GATAa-binding sites. Possible role of the octamer motif shared by the animal hemisphere enhancers The MEME software identified the octamer motif shared by the E1 and T1 enhancers. Point mutations of the motif resulted in severe reduction of enhancer activity. There are at least three possible explanations of how the octamer motif contributes to transcriptional activation. First, Ci-GATAa binds to the motif and activates transcription. The affinity of the GATA sequence for Ci-GATAa is affected by the adjacent sequence, where AGGG facilitates the binding of Ci-GATAa to the
GATA sequence. Second, the octamer motif is a binding site for a transcription factor other than Ci-GATAa. Although E-M1 and T-M1 can bind Ci-GATAa in vitro, these sequences may be occupied in vivo by another transcription factor that recognizes the octamer motif as a whole. Third, Ci-GATAa binds to the GATA part of the octamer motif, while another transcription factor synergistically binds to the AGGG part. The combinatorial binding of two transcription factors may accelerate transcriptional activation. Mutations in E-M1 and T-M1 (GtaAAGGG or GATActtt) indeed affected the binding of Ci-GATAa, supporting the first possibility. However, this does not exclude the second and third explanations. The 397-bp upstream region of Ci-ephrin-Ad contains two strong Ci-GATAa-binding sites (E-G1 and E-G2). However, only E-G2 is necessary, while E-G1 is dispensable for transcriptional activation. Disruption of E-G1 revealed that only a single strong Ci-GATAa-binding site (and a few weak sites?) was sufficient for transcriptional
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sites remained. These observations suggest that the octamer motif is not just one of many Ci-GATAa-binding sites. In vitro binding of Ci-GATAa to the octamer sequence does not necessarily mean that the octamer is also legitimately occupied in vivo by Ci-GATAa. Maternal Ci-GATAa mRNA is ubiquitous, and seems to be translated in both animal and vegetal blastomeres (Bertrand et al. 2003). Disruption of the b-catenin function caused ectopic expression of the Ci-fog reporter gene in the vegetal hemisphere, suggesting that b-catenin suppresses Ci-GATAa at the post-transla€cher et al. 2007). As described tional level (Rothba above, one of the critical GATA sequences within the Ci-fog enhancer is similar to the octamer motif. It is therefore possible that the octamer motif is also involved in the animal-specific and b-catenin-sensitive transcriptional activation. Extensive binding analyses using nuclear extracts and identification of binding proteins will help elucidate what binds to the octamer motif in vivo.
Fig. 6. Effect of point mutations in putative transcription factorbinding sites in the 5´ flanking region of Ci-TGFb-NA1. Diagrams of trangenes containing mutations and their pattern of expression are shown side-by-side. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (a) Tgfna204Z, carrying no mutation. (b) Tgfna (G3 m) Z, carrying a mutation in the GATA sequence T-G3, which overlaps with the octamer motif T-M1. (c) Tgfna (M1 m) Z, carrying a mutation in T-M1. The mutation was created to convert GATAAGGG into GATATCCC, not to disrupt T-G3.
Other transcription factors
activation. In contrast, the Ci-TGFb-NA1 upstream region contained four binding sites for Ci-GATAa. Disruption of only one of them (T-G3 or T-M1) abolished the enhancer activity, although three strong binding
In sea urchin embryos, maternally provided SoxB1 is responsible for gene expression in the presumptive ectodermal region in the animal hemisphere (Kenny et al. 1999). Sox transcription factors are expressed mainly in the central nervous system in vertebrates
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Fig. 7. Electrophoretic mobility shift assay. Double-stranded radioactive probes were incubated with in vitro translated Ci-GATAa protein. For lane 1, TC14-3 was used as a control protein. TC14-3 is a calcium-dependent galactose-binding lectin, whose cDNA was obtained from the budding ascidian Polyandrocarpa misakiensis (Matsumoto et al. 2001). DNA/protein complexes were fractionated on a polyacrylamide gel. The black arrowhead indicates specific signals of the complex’s formation. The open arrowhead indicates non-specific signals. Probes used are indicated below the gel image. Lower case letters (in red) indicate mutagenized nucleotides. (a) The GATA sequences within the Ci-ephrinAd upstream region were examined. (b) The GATA sequences within the CiTGFb-NA1 upstream region were examined. ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
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(Pevny & Lovell-Badge 1997). In the Ciona embryo, SoxF is specifically expressed in the animal hemisphere from the 16-cell stage (Imai et al. 2004). SoxB1 is also expressed in all of the eight animal blastomeres, although it is expressed in anterior vegetal blastomeres (Imai et al. 2004). Therefore, these Sox factors may play a role in the gene regulatory network in the animal hemisphere. In the present study, we found two putative Sox-binding sequences in the 5′ flanking region of Ci-ephrin-Ad. However, these sites were unnecessary for transcriptional activation, at least at the 16-cell stage. Although animal hemisphere-specific expression of many transcription factors, signaling proteins and receptors is observed at the 16-cell stage, these proteins require a time-lag to exhibit their function. Since zygotic expression at the 16-cell stage is the earliest, maternal transcription factors likely play important roles. A large amount of expressed sequence tag (EST) and in situ data have been accumulated for maternal transcripts (e.g. Nishikata et al. 2001). In contrast, information about maternal proteins is still limited (Nomura et al. 2009; Endo et al. 2011). In addition to analyzing enhancers, it is important to characterize maternal proteins involved in activation of the gene regulatory network at the earliest stage of embryogenesis.
Acknowledgments This work was supported in part by Grants-in-aid from the Japan Society for the Promotion of Science (#22570214 and #25440192). We thank Kazuko Hirayama, Chikako Imaizumi, Shota Chiba, Nori Satoh, Yutaka Satou and the Maizuru Fisheries Research Station of Kyoto University for providing us with animals through the National Bio-Resource Project, MEXT, Japan. We are particularly grateful to the late Ms. Kazuko Hirayama who helped us set up the culture system in Kochi University. We also thank Kaz Nitta for sharing his unpublished data.
References Bailey, T. L. & Elkan, C. 1994. Fitting a Mixture Model by Expectation Maximization to Discover Motifs in Biopolymers. Proc. 2nd Int. Conf. Intell. Syst. Mol. Biol. Menlo Park, CA: AAAI Press, pp. 28–36. Bertrand, V., Hudson, C., Caillol, D., Popovici, C. & Lemaire, P. 2003. Neural tissue in ascidian embryos is induced by FGF9/16/20, acting via a combination of maternal GATA and Ets transcription factors. Cell 115, 615–627. Conklin, E. G. 1905. The organization and cell lineage of ascidian egg. J. Acad. Nat. Sci. (Philadelphia) 13, 1–119. Corbo, J. C., Levine, M. & Zeller, R. W. 1997. Characterization of a notochord-specific enhancer from the Brachyury promoter
region of the ascidian, Ciona intestinalis. Development 124, 589–602. Endo, T., Ueno, K., Yonezawa, K., Mineta, K., Hotta, K., Satou, Y., Yamada, L., Ogasawara, M., Takahashi, H., Nakajima, A., Nakachi, M., Nomura, M., Yaguchi, J., Sasakura, Y., Yamazaki, C., Sera, M., Yoshizawa, A. C., Imanishi, T., Taniguchi, H. & Inaba, K. 2011. CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of largescale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality. Nucleic Acids Res. 39 (Database Issue), D807–D814. Fujiwara, S., Corbo, J. C. & Levine, M. 1998. The Snail repressor establishes a muscle/notochord boundary in the Ciona embryo. Development 125, 2511–2520. Hamaguchi, M., Fujie, M., Noda, T. & Satoh, N. 2007. Microarray analysis of zygotic expression of transcription factor genes and cell signaling molecule genes in early Ciona intestinalis embryos. Dev. Growth Differ. 49, 27–37. Imai, K. S., Hino, K., Yagi, K., Satoh, N. & Satou, Y. 2004. Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. Development 131, 4047– 4058. Kanda, M., Wada, H. & Fujiwara, S. 2009. Epidermal expression of Hox1 is directly activated by retinoic acid in the Ciona intestinalis embryo. Dev. Biol. 335, 454–463. Kenny, A. P., Kozlowski, D. J., Oleksyn, D. W., Angerer, L. M. & Angerer, R. C. 1999. SpSoxB1, a maternally encoded transcription factor asymmetrically distributed among early sea urchin blastomeres. Development 126, 5473–5483. Lemaire, P., Smith, W. C. & Nishida, H. 2008. Ascidians and the plasticity of the chordate developmental program. Curr. Biol. 18, R620–R630. Matsumoto, J., Nakamoto, C., Fujiwara, S., Yubisui, T. & Kawamura, K. 2001. A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates. Development 128, 3339–3347. Mertin, S., McDowall, S. G. & Harley, V. R. 1999. The DNA-binding specificity of SOX9 and other SOX proteins. Nucleic Acids Res. 27, 1359–1364. Nishida, H. 1987. Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev. Biol. 121, 526–541. Nishikata, T., Yamada, L., Mochizuki, Y., Satou, Y., Shin-i, T., Kohara, Y. & Satoh, N. 2001. Profiles of maternally expressed genes in fertilized eggs of Ciona intestinalis. Dev. Biol. 238, 315–331. Noda, T. & Satoh, N. 2008. A comprehensive survey of cadherin superfamily gene expression patterns in Ciona intestinalis. Gene Expr. Patterns 8, 349–356. Nomura, M., Nakajima, A. & Inaba, K. 2009. Proteomic profiles of embryonic development in the ascidian Ciona intestinalis. Dev. Biol. 325, 468–481. Pevny, L. H. & Lovell-Badge, R. 1997. Sox genes find their feet. Curr. Opin. Genet. Dev. 7, 338–344. Picco, V., Hudson, C. & Yasuo, H. 2007. Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos. Development 134, 1491–1497. Qiao, Y., Zhu, Y., Sheng, N., Chen, J., Tao, R., Zhu, Q., Zhang, T., Qian, C. & Jing, N. 2012. AP2c regulates neural and epidermal development downstream of the BMP pathway at early stages of ectodermal patterning. Cell Res. 22, 1546–1561.
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€cher, U., Bertrand, V., Lamy, C. & Lemaire, P. 2007. A Rothba combinatorial code of maternal GATA, Ets and b-cateninTCF transcription factors specifies and patterns the early ascidian ectoderm. Development 134, 4023–4032. Satou, Y., Kawashima, T., Shoguchi, E., Nakayama, A. & Satoh, N. 2005. An integrated database of the ascidian, Ciona intestinalis: towards functional genomics. Zool. Sci. 22, 837– 843. €hle, U. & Scholpp, S. 2013. Neurogenesis in zeSchmidt, R., Stra brafish - from embryo to adult. Neural Dev. 8, 3. Stern, C. D. 2005. Neural induction: 10 years on since the ‘default model’. Curr. Opin. Cell Biol. 18, 692–697. Tassy, O., Dauga, D., Daian, F., Sobral, D., Robin, F., Khoueiry, P., Salgado, D., Fox, V., Caillol, D., Schiappa, R., Laporte, B., Rios, A., Luxardi, G., Kusakabe, T., Joly, J. S., Darras, S., Christiaen, L., Contensin, M., Auger, H., Lamy, C., Hud€cher, U., Gilchrist, M., Makabe, K. W., Hotta, son, C., Rothba K., Fujiwara, S., Satoh, N., Satou, Y. & Lemaire, P. 2010. The ANISEED database: digital representation, formalization
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site: Table S1. Primers used for construction of plasmids.
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Develop. Growth Differ. (2013) 55, 776–785
doi: 10.1111/dgd.12100
Original Article
Transcriptional regulation in the early ectodermal lineage of ascidian embryos Yosuke Horikawa, Haruka Matsumoto, Fumika Yamaguchi, † Satomi Ishida and Shigeki Fujiwara* Department of Applied Science, Kochi University, 2-5-1 Akebono-cho, Kochi-shi, Kochi, 780-8520, Japan
In ascidian embryos, ectodermal tissues derive from blastomeres in the animal hemisphere. The animal hemisphere-specific gene expression is observed as early as the 16-cell stage. Here, we characterized animal hemisphere-specific enhancers of three genes, Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4. Deletion analyses identified minimal essential elements. Although these elements contained multiple GATA sequences, electrophoretic mobility shift assays revealed that only some of them were strong binding sites for the transcription factor Ci-GATAa. On the other hand, the motif-searching software MEME identified an octamer, GA (T/G) AAGGG, shared by these enhancers. In Ci-ephrin-Ad and Ci-TGFb-NA1, the octamer was GATAAGGG, which strongly bound Ci-GATAa. The 397-bp upstream region of Ci-ephrin-Ad contained two strong Ci-GATAa-binding sites, one of which was the octamer motif. Mutation in the octamer motif, but not the other Ci-GATAa-binding site, severely affected the enhancer activity. The 204-bp upstream region of Ci-TGFb-NA1 contained four strong Ci-GATAa-binding sites, including the octamer motif. Mutation only in the octamer motif, leaving the other three Ci-GATAa-binding sites intact, abolished the enhancer activity. These results suggest a crucial role for the octamer motif. Key words: ascidian, ectoderm, enhancer, GATA, transcriptional activation.
Introduction The ectoderm arises from the animal hemisphere in chordates, including amphibians and ascidians (Lemaire et al. 2008). It is subsequently subdivided into the epidermis and central nervous system. The mechanism of neural induction has been extensively studied (for reviews see Stern 2005; Schmidt et al. 2013). Factors promoting epidermal specification and suppressing neural fate have also been identified, which are activated by bone morphogenetic protein (e.g. Qiao et al. 2012; Trıbulo et al. 2012). However, few genes have been identified that were expressed in the uncommitted general ectoderm. Our knowledge about *Author to whom all correspondence should be addressed. Email: [email protected] † Present address: Laboratory of Molecular Biology, Medical Research Center, Kochi Medical School, Kochi, 783-8505, Japan. Received 3 September 2012; revised 9 October 2013; accepted 10 October 2013. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists
the mechanisms of specification of the epidermis and general ectoderm is fragmentary. The third cleavage of ascidian embryos divides the embryo into four animal and four vegetal blastomeres. The animal blastomeres mainly give rise to the epidermis and central nervous system (Conklin 1905; Nishida 1987). Nishikata et al. (2001) reported that mRNAs of a few genes were predominantly localized in the animal blastomeres of the 8-cell embryo, although it is not clear whether this localization is achieved by specific zygotic expression or asymmetrical distribution of maternal mRNA. The mRNAs localized to the animal blastomeres include those encoding a WD repeat-containing protein, an F-actin-capping protein, a calcineurin inhibitor, and a putative GTP-binding protein (Nishikata et al. 2001). It is unclear whether these proteins are involved in the ectodermal genetic regulatory cascade. At the 16-cell stage, many genes are expressed exclusively in the animal hemisphere. These include the genes encoding transcription factors; Ci-SoxF €cher et al. 2007). (Imai et al. 2004) and Ci-fog (Rothba Components of cell–cell signaling pathways are also expressed in the animal hemisphere; Ci-ephrin-Aa, Ci-ephrin-Ad, Ci-TGFb-NA1, Ci-Smad1/5 and Ci-Fz4
Early ectodermal enhancers in ascidians
(Imai et al. 2004; Hamaguchi et al. 2007; Picco et al. 2007). Cell adhesion molecules (Ci-d1-protocadherinlike and Ci-d-protocadherin-4) are also specifically expressed (Noda & Satoh 2008). Transcription of Ci-fog in the animal hemisphere is activated by the transcription factor Ci-GATAa €cher et al. 2007). A Ci-GATAa-specific mor(Rothba pholino oligo suppresses the enhancer activity of €cher et al. 2007). Transcriptional activaCi-fog (Rothba tion of Ci-otx in the animal hemisphere also requires Ci-GATAa (Bertrand et al. 2003). The enhancer regions of these genes contain multiple GATA sequences (Ber€cher et al. 2007), although trand et al. 2003; Rothba binding of the Ci-GATAa protein to these sequences was not demonstrated. In addition, it is not yet clear whether only Ci-GATAa is responsible for activation of animal hemisphere-specific genes. In the present study, we identified animal hemispherespecific enhancer elements within the 5′ flanking regions of three genes (Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4). These genes were chosen because their specific expression was clear and strong (Imai et al. 2004; Picco et al. 2007). We found multiple GATA sequences in the upstream regions of these genes. Electrophoretic mobility shift assays revealed that some, but not all, of them strongly bind the Ci-GATAa protein. However, disruption of only one of them, whose GATA sequence was followed by AGGG, severely affected the enhancer activity. Based on these results, the possible involvement of transcription factor (s) other than Ci-GATAa is discussed.
Materials and methods Animals Juvenile adults of Ciona intestinalis were purchased from the National Bio-Resource Project of MEXT, Japan. The animals were cultured in Tosa Bay near the Usa Marine Biological Institute of Kochi University. Eggs and sperm were obtained from the gonoducts of mature adults. Eggs were inseminated with non-self sperm. Fertilized eggs were dechorionated with 0.05% actinase E (Kaken Pharmaceutical) and 1% sodium thioglycolate. Embryos were reared in artificial seawater Super Marine Art SF1 (Tomita Pharmaceutical). Construction of plasmids Genomic DNA fragments corresponding to the promoter region of Ci-ephrin-Ad, Ci-TGFb-NA1 and CiFz4 were amplified by polymerase chain reaction (PCR), using the primers ephrin1.7F and ephrin1.7R for Ci-ephrin-Ad, tgfna3.0F and tgfna3.0R for
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Ci-TGFb-NA1, and fz2.0F and fz2.0R for Ci-Fz4 (Table S1). The PCR products were inserted into pGEM-T (Promega). The Ci-ephrin-Ad fragment was excised with PstI and BamHI from pGEM-T, while the Ci-TGFb-NA1 and Ci-Fz4 fragments were excised with XhoI and BamHI. These fragments were inserted into pSP72-1.27 (Corbo et al. 1997). The resultant plasmids were named Ephrin1648Z, Tgfna2940Z and Fz1964Z, respectively. For construction of Ephrin545Z, Ephrin497Z, Ephrin447Z, Ephrin397Z and Ephrin333Z, PCR was carried out using a lacZ-specific primer (gal-A2) and a Ci-ephrin-Ad-specific primer (ephrin545F, ephrin497F, ephrin447F, ephrin397F and ephrin333F, respectively; Table S1). Ephrin1648Z was used as a template. For construction of Tgfna799Z, Tgfna599Z, Tgfna399Z, Tgfna204Z and Tgfna66Z, PCR was carried out using tgfna3.0R and one of the following primers (tgfna799F, tgfna599F, tgfna399F, tgfna204F and tgfna66F, respectively; Table S1). Tgfna2940Z was used as a template. For construction of Fz1425Z, Fz1294Z, Fz975Z, Fz784Z and Fz387Z, PCR was carried out using fz2.0R and one of the following primers (fz1425F, fz1294F, fz975F, fz784F and fz387F, respectively; Table S1). Fz1964Z was used as a template. PCR products were once inserted into pGEM-T, excised with XhoI and BamHI, and inserted into pSP72-1.27. Site-directed mutagenesis was performed according to the protocol described by Fujiwara et al. (1998). To create point mutations within the Ci-ephrin-Ad enhancer, the XhoI-BamHI fragment of Ephrin397Z was inserted into pBluescript II SK+ (Stratagene). Uracilcontaining single-stranded DNA was prepared using the Escherichia coli strain CJ236 (Takara). Mutagenic oligonucleotides used were E-S1 m, E-S2 m, E-G1 m, E-G2 m, E-S1G1 m and E-Mm (Table S1). For point mutations in the Ci-TGFb-NA1 enhancer, we transformed CJ236 with pGEM-T containing the PCR product using primers tgfna204F and tgfna3.0R (described above). Mutagenic oligonucleotides used were T-Gm and T-Mm (Table S1). Electroporation and detection of transgene expression Plasmid DNA was prepared using QIAGEN tip-100 (Qiagen). Transgenes were introduced into dechorionated embryos by electroporation, according to the protocol described by Corbo et al. (1997). Expression of lacZ was detected by in situ hybridization as described by Kanda et al. (2009). In each experiment, 100–400 electroporated embryos were observed under an AZ100 microscope (Nikon). Embryos expressing lacZ in one or more blastomeres were counted as positive.
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The percentage of positive embryos among morphologically normal embryos was calculated. Electrophoretic mobility shift assay The entire translated region of Ci-GATAa was amplified by PCR using the cDNA clone “cien229100” as a template (Tassy et al. 2010; http://www.aniseed.cnrs.fr/ index.php). The PCR was carried out using primers GATA-F and GATA-Nhe-R (Table S1), and Tks Gflex DNA polymerase (Takara), according to the protocol supplied by the manufacturer. The product of the PCR was briefly treated with Taq DNA polymerase (Ampliqon) to create the 3′ overhang of a single adenine nucleotide, and inserted into pGEM-T. The Ci-GATAa cDNA was again amplified using Tks Gflex DNA polymerase and the primers GATA-F and M13-Forward-40 (5′-GTTTTCCCAGTCACGAC-3′). The product of the PCR was excised with NheI. The pCMX-hRARa plasmid (Umesono et al. 1991) was digested with MscI and NheI to remove the cDNA encoding human retinoic acid receptor a (hRARa). The Ci-GATAa cDNA was inserted into the pCMX vector. The Ci-GATAa protein was produced using the TNT coupled reticulocyte lysate system (Promega). Double-stranded DNA
probes were labeled with [c-32P] adenosine triphosphate (Perkin Elmer Japan), according to the protocol described by Kanda et al. (2009). Electrophoretic mobility shift assays (EMSAs) were carried out using a Gel-shift assay system (Promega). Autoradiograms were obtained using BAS2500 (Fuji Film).
Results Identification of early animal-hemisphere enhancers A fragment of the C. intestinalis genomic DNA was obtained, which contained the promoter region of Ciephrin-Ad, including putative transcription and translation initiation sites (Fig. 1a). The fragment was fused in-frame with lacZ and introduced into fertilized eggs by electroporation (Fig. 1b). The resultant transgene, named Ephrin1648Z, contained 1648 bp of the 5′ flanking region, according to the gene model KH.C3.716.v1.A.SL1-1 in the C. intestinalis genome database (Satou et al. 2005; http://ghost.zool.kyoto-u. ac.jp/SearchGenomekh.html). Ephrin1648Z was introduced into one-cell embryos. The embryos were fixed at the 16-cell stage and the lacZ mRNA was detected by in situ hybridization. Ephrin1648Z was expressed in
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Fig. 1. The animal hemisphere enhancer of Ci-ephrin-Ad. (a) The genomic structure of the Ci-ephrin-Ad gene, predicted in the Ciona intestinalis genome database (Satou et al. 2005; http://ghost.zool.kyoto-u.ac.jp/SearchGenomekh.html). Exons are indicated by boxes. The open reading frame is in green. The 1.6-kb upstream region examined in the present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The E1 enhancer is indicated by a red box. Percentage of embryos expressing lacZ among normally developing embryos is indicated. (c–h) The expression pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up. Names of the blastomeres are indicated in (c). ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
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all of the blastomeres (a5.3, a5.4, b5.3 and b5.4 blastomere pairs) in the animal hemisphere (Fig. 1c). Transgenes containing 545, 497, 447, or 397 bp of the upstream sequence gave essentially similar patterns of expression, although some embryos did not express lacZ in all of the eight blastomeres (Fig. 1d–g). No embryo carrying Ephrin333Z expressed lacZ in any blastomere (Fig. 1h). These results indicate that 397 bp of the 5′ flanking region of Ci-ephrin-Ad is sufficient, and the region between 397 and 334, named E1, is necessary for transcriptional activation in the animal hemisphere. We also isolated the promoter regions of Ci-TGFb-NA1 and Ci-Fz4, and placed them upstream of lacZ. (Figs 2 and 3). A transgene named Tgfna2940Z contained 2940 bp of the 5′ flanking region of Ci-TGFb-NA1, according to the gene model KH.C4.547.v1.A.nonSL1-1 (Fig. 2a,b). Tgfna2940Z was expressed in all of the eight blastomeres in the animal hemisphere of the 16-cell embryo (Fig. 2c). Deletion analyses revealed that 204 bp of the upstream sequence of Ci-TGFbNA1 was sufficient, and the region between 204 and 66, named T1, was necessary for transcriptional activation in the animal hemisphere (Fig. 2d–h). Fz1964Z contained 1964 bp of the 5′ flanking region of Ci-Fz4, according to the gene model KH.C6.162.v2.A. SL2-1 (Fig. 3a,b). Fz1964Z recapitulated the animal (a)
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hemisphere-specific expression of Ci-Fz4 (Fig. 3c). Deletion analyses revealed that 975 bp of the upstream sequence of Ci-Fz4 was sufficient, and the region between 975 and 784, named F1, was necessary for transcriptional activation in the animal hemisphere (Fig. 3d–h). The region between 783 and 67 was deleted from Fz975Z. The resultant transgene was not expressed, suggesting that the proximal region (downstream to 784) also contains enhancer element (s) necessary for transcriptional activation (data not shown). Possible binding sites for transcription factors We searched for sequence motifs shared by Ci-ephrin-Ad, Ci-TGFb-NA1 and Ci-Fz4, using MEME (Bailey & Elkan 1994; http://meme.sdsc.edu/meme/intro.html). MEME identified an octamer sequence, GA (T/G) AAGGG. The E1 and T1 enhancers contained a single octamer motif each (GATAAGGG), named E-M1 and T-M1, respectively (Fig. 4). MEME did not find this motif within the 975-bp proximal region of Ci-Fz4. However, we found a similar sequence, GAAAAGGG, at nucleotide position 73 of Ci-Fz4 (data not shown). We also searched for putative binding sites for known transcription factors. Ci-GATAa is necessary for activation of genes in the animal hemisphere of the Ciona (c)
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Fig. 2. The animal hemisphere enhancer of Ci-TGFb-NA1. (a) The genomic structure of the Ci-TGFb-NA1 gene, predicted in the Ciona intestinalis genome database. Exons are indicated by boxes. The open reading frame is in green. The 2.94-kb upstream region examined in the present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The T1 enhancer is indicated by a red box. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (c–h) The expression pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up. ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
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Fig. 3. The animal hemisphere enhancer of Ci-Fz4. (a) The genomic structure of the Ci-Fz4 gene, predicted in the Ciona intestinalis genome database. Exons are indicated by boxes. The open reading frame is in green. The 1.96-kb upstream region examined in the present study is indicated. (b) Diagrams showing the different 5´ flanking regions used for the analysis in (c–h). The F1 enhancer is indicated by a red box. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (c–h) The expression pattern of transgenes shown in (b). All panels show the animal side of the 16-cell embryos. The anterior side is up.
€cher et al. 2007). The binding embryo (Rothba sequence for Ci-GATAa is 5′-GATA-3′, and it had no strong constraint on surrounding sequences (K. Nitta & P. Lemaire, pers. comm., 2012). Ci-SoxF is the only transcription factor whose mRNA is exclusively detected in the animal hemisphere at the 16-cell stage (Imai et al. 2004). The core recognition sequence for the Sox group transcription factors is AACAAT (Mertin et al. 1999). In vitro binding experiments revealed that the consensus sequence for C. intestinalis Sox proteins is (A/G) (A/C) CAA (T/A) (K. Nitta & P. Lemaire, pers. comm., 2012). We found six GATA-binding sites and two Sox-binding sites within the 397-bp proximal region of Ci-ephrin-Ad (Fig. 4a). The E1 enhancer contained two GATA-binding sites (named E-G1 and EG2) and one Sox-binding site (named E-S1; Fig. 4a,b). The 204-bp upstream region of Ci-TGFb-NA1 contained no Sox-binding site, but four GATA-binding sites (Fig. 4a). The T1 enhancer contained three of them (named T-G1–T-G3; Fig. 4c). E-M1 and T-M1 included the GATA sequence (E-G2 and T-G3, respectively, Fig. 4). The octamer motif is necessary for enhancer activity Since the E1 enhancer is necessary for transcriptional activation in the animal hemisphere, we tested the
requirement of putative binding sites for transcription factors by site-directed mutagenesis. First, a point mutation was generated at the Sox-binding site within the E1 enhancer (E-S1; Fig. 5a,b). The mutant transgene, named Ephrin (S1 m) Z, was expressed normally in the animal hemisphere at the 16-cell stage (Fig. 5b). Another possible Sox-binding sequence was found downstream of nucleotide position 333 (E-S2; Fig. 5a). Disruption of E-S2, or a double mutation in E-S1 and E-S2, did not affect the expression in the animal hemisphere (Fig. 5c,d). One of the two GATAbinding sites within the E1 enhancer (E-G1) was mutagenized (Fig. 5a,e). The mutant transgene, named Ephrin (G1 m) Z, was expressed in the animal hemisphere (Fig. 5e). In contrast, a mutation in the other GATA sequence (E-G2) caused complete silencing of the reporter gene (Fig. 5f). Note that E-G2 overlaps with E-M1 (GATAAGGG) (Figs. 4b and 5a), and that a point mutation in E-G2 completely inactivated the enhancer that still contained five additional GATA sequences (see Fig. 4a). We then introduced a point mutation into E-M1 without disrupting E-G2 (GATAAGGG to GATActtt). The resultant transgene, named Ephrin (M1 m) Z, was not expressed in any blastomere (Fig. 5g). About 83% of the embryos carrying Ephrin397Z expressed lacZ, while only 14% of the embryos carrying Ephrin (M1 m) Z expressed it.
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DNA/protein complex (Fig. 7a, lane 2). We prepared a mutant probe (E-M1 m), in which AGGG of E-M1 was disrupted without affecting GATA. E-M1m did not form a complex with Ci-GATAa (Fig. 7a, lane 3). A probe containing E-G1 formed a complex with Ci-GATAa (Fig. 7a, lane 4). E-G3, E-G5 and E-G6 formed a weak band (Fig. 7a, lanes 5, 7, 8). No specific shift was observed with E-G4 (Fig. 7a, lanes 6). A probe containing T-M1 (overlapping with T-G3) and T-G2 formed a complex with Ci-GATAa (Fig. 7b, lane 2). The shifted band became weak when mutation was created in either T-G2 or T-G3 (Fig. 7b, lanes 3, 5). When the AGGG moiety of T-M1 was disrupted without affecting GATA, the complex’s formation was also affected (Fig. 7b, lane 4). No shifted band was observed when both T-G2 and T-G3 were mutated (Fig. 7b, lane 6). T-G1 and T-G4 also strongly formed a complex with Ci-GATAa (Fig. 7b, lanes 7, 8).
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Discussion Binding of Ci-GATAa is strongly influenced by flanking sequences
Fig. 4. Putative transcription factor-binding sites in the E1 and T1 enhancers (a) Schematic diagrams showing the 5´ flanking regions of Ci-ephrin-Ad and Ci-TGFb-NA1. Putative binding sites for the GATA and Sox transcription factors are indicated by blue and yellow boxes, respectively. The octamer motif (GATAAGGG) is indicated by red boxes. (b) Nucleotide sequence of the E1 enhancer. (c) Nucleotide sequence of the T1 enhancer.
The T1 enhancer contains three GATA sequences, one of which (T-G3) overlaps with T-M1 (GATAAGGG) (Figs 4c and 6a). Disruption of T-G3 (GATAAGGG to GtaAAGGG) completely silenced the expression of lacZ (Fig. 6b). A point mutation of T-M1 that did not disrupt T-G3 (GATAAGGG to GATActtt) also caused severe inactivation of the enhancer (Fig. 6c). About 81% of the embryos carrying Tgfna204Z expressed lacZ, while 3.6% of the embryos carrying Tgfna (M1 m) Z did so. Binding of the Ci-GATAa protein to wild-type and mutagenized DNA sequences Electrophoretic mobility shift assays were carried out using a full-length Ci-GAGAa protein synthesized in vitro. An oligonucleotide probe containing the E-M1 (GATAAGGG) and its flanking sequences formed a
Direct binding assays of the ascidian GATA protein were first reported in the present study. Our EMSA revealed cases in which the affinity of the Ci-GATAa binding to DNA was strongly affected by the sequences surrounding the core GATA sequence. The 397-bp upstream region of Ci-ephrin-Ad contains six GATA sequences. However, only two of them (E-G1 and E-G2) can strongly bind Ci-GATAa. These results seem inconsistent with unpublished results obtained by K. Nitta and P. Lemaire (pers. comm., 2012). They searched for a consensus Ci-GATAa-binding sequence by the systematic evolution of ligands using exponential enrichment (SELEX). They did not see a strong preference outside of the GATA sequence, although TGATAA was a little better (K. Nitta & P. Lemaire, pers. comm., 2012). In the present study, Ci-GATAa seemed to prefer GATAAGGG. The underlined nucleotides may not have been recognized by SELEX, possibly because only two or three of the underlined nucleotides are conserved in many cases. €cher et al. (2007) Bertrand et al. (2003) and Rothba carried out mutagenesis of many GATA sequences within the enhancer region of Ci-otx and Ci-fog, and reported strong suppression of reporter genes. Similar results were obtained by antisense-mediated knock€cher down of Ci-GATAa (Bertrand et al. 2003; Rothba et al. 2007). Two critical GATA sequences within the Ci-fog enhancer are GATAAAGT and GATACTTT €cher et al. 2007). The former is similar to the (Rothba octamer motif, but the latter looks like the mutant
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(g) Fig. 5. Effect of point mutations in putative transcription factor-binding sites in the 5´ flanking region of Ci-ephrin-Ad. Diagrams of transgenes and their pattern of expression are shown side-by-side. Percentage of the embryos expressing lacZ among normally developing embryos is also indicated. (a) Ephrin397Z, carrying no mutation. (b) Ephrin (S1 m) Z, carrying a mutation in the Sox-binding site ES1. (c) Ephrin (S2 m) Z, carrying a mutation in the other Sox-binding site E-S2. (d) Ephrin (S1S2 m) Z, carrying mutations in both E-S1 and E-S2. (e) Ephrin (G1 m) Z, carrying a mutation in the GATA sequence E-G1. (f) Ephrin (G2 m) Z, carrying a mutation in the GATA sequence E-G2, which overlaps with the octamer motif E-M1. (g) Ephrin (M1 m) Z, carrying a mutation in E-M1. The mutation was created to convert GATAAGGG into GATATCCC, not to disrupt E-G2.
sequences that did not bind Ci-GATAa in the present study. None of the seven GATA sequences within the Ci-otx enhancer look similar to the strong binding sites (Bertrand et al. 2003). Considering the present results, direct binding assays will be necessary to confirm that these GATA sequences are genuine Ci-GATAa-binding sites. Possible role of the octamer motif shared by the animal hemisphere enhancers The MEME software identified the octamer motif shared by the E1 and T1 enhancers. Point mutations of the motif resulted in severe reduction of enhancer activity. There are at least three possible explanations of how the octamer motif contributes to transcriptional activation. First, Ci-GATAa binds to the motif and activates transcription. The affinity of the GATA sequence for Ci-GATAa is affected by the adjacent sequence, where AGGG facilitates the binding of Ci-GATAa to the
GATA sequence. Second, the octamer motif is a binding site for a transcription factor other than Ci-GATAa. Although E-M1 and T-M1 can bind Ci-GATAa in vitro, these sequences may be occupied in vivo by another transcription factor that recognizes the octamer motif as a whole. Third, Ci-GATAa binds to the GATA part of the octamer motif, while another transcription factor synergistically binds to the AGGG part. The combinatorial binding of two transcription factors may accelerate transcriptional activation. Mutations in E-M1 and T-M1 (GtaAAGGG or GATActtt) indeed affected the binding of Ci-GATAa, supporting the first possibility. However, this does not exclude the second and third explanations. The 397-bp upstream region of Ci-ephrin-Ad contains two strong Ci-GATAa-binding sites (E-G1 and E-G2). However, only E-G2 is necessary, while E-G1 is dispensable for transcriptional activation. Disruption of E-G1 revealed that only a single strong Ci-GATAa-binding site (and a few weak sites?) was sufficient for transcriptional
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sites remained. These observations suggest that the octamer motif is not just one of many Ci-GATAa-binding sites. In vitro binding of Ci-GATAa to the octamer sequence does not necessarily mean that the octamer is also legitimately occupied in vivo by Ci-GATAa. Maternal Ci-GATAa mRNA is ubiquitous, and seems to be translated in both animal and vegetal blastomeres (Bertrand et al. 2003). Disruption of the b-catenin function caused ectopic expression of the Ci-fog reporter gene in the vegetal hemisphere, suggesting that b-catenin suppresses Ci-GATAa at the post-transla€cher et al. 2007). As described tional level (Rothba above, one of the critical GATA sequences within the Ci-fog enhancer is similar to the octamer motif. It is therefore possible that the octamer motif is also involved in the animal-specific and b-catenin-sensitive transcriptional activation. Extensive binding analyses using nuclear extracts and identification of binding proteins will help elucidate what binds to the octamer motif in vivo.
Fig. 6. Effect of point mutations in putative transcription factorbinding sites in the 5´ flanking region of Ci-TGFb-NA1. Diagrams of trangenes containing mutations and their pattern of expression are shown side-by-side. Percentage of the embryos expressing lacZ among normally developing embryos is indicated. (a) Tgfna204Z, carrying no mutation. (b) Tgfna (G3 m) Z, carrying a mutation in the GATA sequence T-G3, which overlaps with the octamer motif T-M1. (c) Tgfna (M1 m) Z, carrying a mutation in T-M1. The mutation was created to convert GATAAGGG into GATATCCC, not to disrupt T-G3.
Other transcription factors
activation. In contrast, the Ci-TGFb-NA1 upstream region contained four binding sites for Ci-GATAa. Disruption of only one of them (T-G3 or T-M1) abolished the enhancer activity, although three strong binding
In sea urchin embryos, maternally provided SoxB1 is responsible for gene expression in the presumptive ectodermal region in the animal hemisphere (Kenny et al. 1999). Sox transcription factors are expressed mainly in the central nervous system in vertebrates
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Fig. 7. Electrophoretic mobility shift assay. Double-stranded radioactive probes were incubated with in vitro translated Ci-GATAa protein. For lane 1, TC14-3 was used as a control protein. TC14-3 is a calcium-dependent galactose-binding lectin, whose cDNA was obtained from the budding ascidian Polyandrocarpa misakiensis (Matsumoto et al. 2001). DNA/protein complexes were fractionated on a polyacrylamide gel. The black arrowhead indicates specific signals of the complex’s formation. The open arrowhead indicates non-specific signals. Probes used are indicated below the gel image. Lower case letters (in red) indicate mutagenized nucleotides. (a) The GATA sequences within the Ci-ephrinAd upstream region were examined. (b) The GATA sequences within the CiTGFb-NA1 upstream region were examined. ª 2013 The Authors Development, Growth & Differentiation ª 2013 Japanese Society of Developmental Biologists
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(Pevny & Lovell-Badge 1997). In the Ciona embryo, SoxF is specifically expressed in the animal hemisphere from the 16-cell stage (Imai et al. 2004). SoxB1 is also expressed in all of the eight animal blastomeres, although it is expressed in anterior vegetal blastomeres (Imai et al. 2004). Therefore, these Sox factors may play a role in the gene regulatory network in the animal hemisphere. In the present study, we found two putative Sox-binding sequences in the 5′ flanking region of Ci-ephrin-Ad. However, these sites were unnecessary for transcriptional activation, at least at the 16-cell stage. Although animal hemisphere-specific expression of many transcription factors, signaling proteins and receptors is observed at the 16-cell stage, these proteins require a time-lag to exhibit their function. Since zygotic expression at the 16-cell stage is the earliest, maternal transcription factors likely play important roles. A large amount of expressed sequence tag (EST) and in situ data have been accumulated for maternal transcripts (e.g. Nishikata et al. 2001). In contrast, information about maternal proteins is still limited (Nomura et al. 2009; Endo et al. 2011). In addition to analyzing enhancers, it is important to characterize maternal proteins involved in activation of the gene regulatory network at the earliest stage of embryogenesis.
Acknowledgments This work was supported in part by Grants-in-aid from the Japan Society for the Promotion of Science (#22570214 and #25440192). We thank Kazuko Hirayama, Chikako Imaizumi, Shota Chiba, Nori Satoh, Yutaka Satou and the Maizuru Fisheries Research Station of Kyoto University for providing us with animals through the National Bio-Resource Project, MEXT, Japan. We are particularly grateful to the late Ms. Kazuko Hirayama who helped us set up the culture system in Kochi University. We also thank Kaz Nitta for sharing his unpublished data.
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web-site: Table S1. Primers used for construction of plasmids.
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