C 2014 Wiley Periodicals, Inc. V

genesis 53:170–182 (2015)

TECHNOLOGY REPORT

Application of the cis-Regulatory Region of a Heat-Shock Protein 70 Gene to Heat-Inducible Gene Expression in the Ascidian Ciona intestinalis Akane Kawaguchi,1 Nanami Utsumi,2 Maki Morita,3 Aya Ohya,1 and Shuichi Wada2* 1

Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

2

Department of Animal Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

3

Guraduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

Received 29 April 2014; Revised 28 October 2014; Accepted 29 October 2014

Summary: Temporally controlled induction of gene expression is a useful technique for analyzing gene function. To make such a technique possible in Ciona intestinalis embryos, we employed the cis-regulatory region of the heat-shock protein 70 (HSP70) gene Ci-HSPA1/6/7-like for heat-inducible gene expression in C. intestinalis embryos. We showed that Ci-HSPA1/6/7-like becomes heat shock-inducible by the 32-cell stage during embryogenesis. The 50 -upstream region of Ci-HSPA1/6/7-like, which contains heat-shock elements indispensable for heat-inducible gene expression, induces the heat shockdependent expression of a reporter gene in the whole embryo from the 32-cell to the middle gastrula stages and in progressively restricted areas of embryos in subsequent stages. We assessed the effects of heat-shock treatments in different conditions on the normality of embryos and induction of transgene expression. We evaluated the usefulness of this technique through overexpression experiments on the well-characterized, developmentally relevant gene, Ci-Bra, and showed that this technique is applicable for inferring the gene function C 2014 Wiley in C. intestinalis. genesis 53:170–182, 2015. V Periodicals, Inc.

under the control of a tissue-specific or ubiquitous enhancer (Stolfi and Christiaen, 2012). A limitation of these methods is the difficulty in controlling the timing of misexpression of genes, and conditional gene expression serves as a promising method to overcome this limitation. To facilitate conditional gene expression in C. intestinalis embryos, we focused on a heat-inducible gene expression technique by exploiting the heat-shock response. A suitable gene for the source of the cis-regulatory element in heat-inducible gene expression technique is one that exhibits minimal or no expression under normal temperature and strong expression under heat shock. In C. intestinalis, a gene that meets these requirements is Ci-HSPA1/6/7-like. This gene is the member of the cytoplasmic group of the DnaK subfamily of the heat shock protein 70 (HSP70) family in C. intestinalis (Wada et al., 2006). In the juvenile stage, the expression of Ci-HSPA1/6/7-like is low under normal temperature and is strongly induced by heat-shock treatment (e.g., the treatment at 28 C for 1 h leads to approximately 760-fold induction; Fujikawa et al., 2010). Therefore, in this study, we described the

Key words: HSP70; Ci-HSPA1/6/7-like; cis-regulatory region; Ci-Bra

Experimental misexpression of genes is a major approach for elucidating gene functions. In the ascidian Ciona intestinalis, misexpression experiments have been performed through mRNA injection or electroporationmediated delivery of plasmid DNA containing a cDNA

* Correspondence to: Shuichi Wada, Department of Animal Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan. E-mail: [email protected] Contract grant sponsor: JSPS KAKENHI, Japan; Contract grant number: 20770183 Published online 4 November 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/dvg.22834

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FIG. 1. Heat-shock response of Ci-HSPA1/6/7-like expression in Ciona intestinalis cleavage-stage embryos. (a) Frequency of Ci-HSPA1/ 6/7-like expression in embryos. Eight-, 16-, 32-, or 64-cell stage embryos were heat shocked at 28 C for 30 min or 1 h. In control experiments, embryos were kept at 18 C. Expression of Ci-HSPA1/6/7-like in these embryos was detected by whole mount in situ hybridization. Embryos were ranked in order of the strength and range of Ci-HSPA1/6/7-like expression. The numbers of embryos examined are indicated above the graph. (b) Spatial expression patterns of Ci-HSPA1/6/7-like visualized by whole mount in situ hybridization.

expression pattern of Ci-HSPA1/6/7-like during embryogenesis, characterized the cis-regulatory region of this gene, and employed it for heat-inducible gene expression in C. intestinalis embryos. RESULTS AND DISCUSSION Expression of Ci-HSPA1/6/7-like in Embryos Under Normal and Heat-Shock Conditions Because no report describes the heat-shock response of this gene in C. intestinalis embryos, we first examined the stage when Ci-HSPA1/6/7-like becomes heat shockinducible during embryogenesis. Cleavage stage embryos were heat-shocked at 28 C for 30 min or 1 h and examined for Ci-HSPA1/6/7-like expression by whole mount in situ hybridization. Approximately 20% of embryos heat

shocked at the eight-cell stage for 30 min exhibited CiHSPA1/6/7-like expression (Fig. 1a,b). When heat shock was conducted for 1 h, heat shock from the eight-cell stage caused Ci-HSPA1/6/7-like expression in 50% of the treated embryos (Fig. 1a,b). The rate of embryos showing Ci-HSPA1/6/7-like expression increased to >90% when embryos were heat shocked from the 16-, 32-, or 64-cell stages (Fig. 1a,b). Under the same staining condition, CiHSPA1/6/7-like expression was not detected in control embryos reared at 18 C (Fig. 1a). During cleavage stages, blastomeres divided every 30 min under normal temperature (18 C) and faster at 28 C. These results suggest that Ci-HSPA1/6/7-like becomes heat shock-inducible by the 32-cell stage at the latest and a heat-inducible gene expression technique is valid after this stage. Following this, we examined the expression pattern of Ci-HSPA1/6/7-like under control and heat-shock

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FIG. 2. Heat-shock response of Ci-HSPA1/6/7-like expression in Ciona intestinalis embryos after the middle gastrula stage. Spatial expression patterns of Ci-HSPA1/6/7-like visualized by whole mount in situ hybridization are presented. Embryos at the middle gastrula, middle neurula, early tailbud II, or middle tailbud II stages were heat shocked at 28 C for 1 h and examined for Ci-HSPA1/6/7-like expression. In the control experiments, embryos were kept at 18 C. Staining reactions for whole mount in situ hybridization were performed for 8 h or 20 h. Hybridization with probes for GFP was performed as negative control experiment for whole mount in situ hybridization. Number of embryos examined is shown at the lower right. n.t.: Not tested.

conditions during later stages of development. Embryos at the middle gastrula, middle neurula, early tailbud II, or middle tailbud II stages were heat shocked at 28 C for 1 h and examined for Ci-HSPA1/6/7-like expression by whole mount in situ hybridization. Hundred percent of the embryos showed Ci-HSPA1/6/7-like expression irrespective of the timing of heat shock. Embryos heat shocked at the middle gastrula stage showed strong and uniform expression of Ci-HSPA1/6/7-like (Fig. 2). In embryos heat shocked at the middle neurula stage, CiHSPA1/6/7-like expression was also detected in the whole body and was stronger in the trunk than in the tail (Fig. 2). Heat-shock treatment at the early tailbud II stage induced the expression of Ci-HSPA1/6/7-like in every part of the body, although the expression was weaker in the epidermis than in the other tissues (Fig. 2). In embryos heat shocked at the middle tailbud II stage, strong and uniform expression of Ci-HSPA1/6/7like was detected (Fig. 2). Under the same staining condition (staining for 8 h), Ci-HSPA1/6/7-like expression

was not detected in the control embryos reared at 18 C (Fig. 2). Longer (20 h) staining showed weak expression of Ci-HSPA1/6/7-like in control embryos (Fig. 2). These results suggest that Ci-HSPA1/6/7-like is heat shock-inducible in the whole body from the middle gastrula through middle tailbud II stages and that weak basal expression of Ci-HSPA1/6/7-like occurs under normal conditions. Expression of a Reporter Gene Under the Control of the cis-Regulatory Region of Ci-HSPA1/6/7-like Next, we tested whether the cis-regulatory region of Ci-HSPA1/6/7-like is usable for driving heat shockdependent transcription of a reporter gene. For this, we generated the plasmid construct that contains a 2,000 bp (relative to the translation initiation site) fragment of the 50 -upstream region of Ci-HSPA1/6/7-like and the LacZ reporter and introduced it into fertilized eggs by electroporation. The eggs were allowed to develop up

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to six different stages, heat shocked at 28 C for 1 h, and examined for b-galactosidase activity by X-gal staining. After approximately 2 h of staining, >95% of heatshocked embryos exhibited X-gal staining irrespective of the timing of the heat-shock treatment (Fig. 3a). Under the same staining condition, few control embryos that were electroporated with the plasmid but were not heat-shocked showed X-gal staining (Fig. 3a). The spatial patterns of the staining in the heat-shocked embryos varied according to the timing of the heatshock treatment. With regard to embryos heat shocked at the 32-cell or middle gastrula stages, X-gal staining was observed in all seven types of tissues examined, with frequencies ranging from 69 to 82% (Fig. 3b), and staining was detected in a broad domain of the embryos (Fig. 3c). When the heat-shock treatment was performed at the middle neurula stage, all types of tissues showed X-gal staining in 81% to 96% of the cases (Fig. 3b); however, staining in the epidermis was restricted to the anterior part of the embryos (Fig. 3c). It was unclear whether the boundary of X-gal staining in the epidermis corresponds to the trunk-tail boundary. The heat-shock treatment at the early tailbud II and the middle tailbud II stages resulted in a decrease in the frequency of the staining in the epidermis to 34% and 10%, respectively (Fig. 3b,c). When embryos were heat shocked at the middle tailbud III stage, the frequency of X-gal staining severely decreased in the mesenchyme, endoderm, and epidermis (27%, 19%, and 17%, respectively) and slightly decreased in the notochord, spinal cord, and sensory vesicle (58%, 63%, and 64%, respectively; Fig. 3b,c). Moreover, the area of X-gal staining in these tissues was small. Therefore, X-gal staining in the muscle was prominent in these embryos (Fig. 3c). As previously described in this report, the expression of endogenous Ci-HSPA1/6/7-like was induced by heat shock in every part of embryos, although the strength of the expression was uneven in embryos heat shocked at the middle neurula or early tailbud II stages (Fig. 2). Therefore, the patterns of X-gal staining in embryos heat shocked after the middle neurula stage differed from the expression patterns of endogenous Ci-HSPA1/6/7-like. The reason for this inconsistency remains unknown and needs to be investigated. However, this phenomenon does not seem to be specific to the cis-regulatory region of Ci-HSPA1/6/7-like because a similar pattern of X-gal staining was observed when the fragment of the 50 -upstream region of Ci-HSPA1/6/7-like in the construct was replaced with that of another heat shock-inducible gene, CiHsp24.4 (Franck et al., 2004; Fig. 4). It is also unlikely that the inconsistency is caused by a harmful influence of the heat-shock treatment because restriction of the X-gal staining region was not observed when the fragment of the 50 -upstream region of Ci-HSPA1/6/7-like in the construct was replaced with that of the nonheat

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shock-inducible, ubiquitously expressed gene Ci-EF1a (Fig. 4). In summary, these results suggest that the gene expression technique exploiting the cis-regulatory region of Ci-HSPA1/6/7-like offers heat shockdependent expression of a given gene in the whole embryo from the 32-cell to the middle gastrula stages and in progressively restricted areas of embryos thereafter. Characterization of the cis-Regulatory Region of Ci-HSPA1/6/7-like When a new experimental technique is introduced, it is important that the mechanism underlying the technique is understood. However, the mechanism of heatshock response has not been investigated in C. intestinalis till date. Therefore, we next characterized the cisregulatory region of Ci-HSPA1/6/7-like to obtain an insight into the mechanism that controls the heat-shock induction of this gene. In eukaryotes, the major regulatory players of heat-shock response are heat-shock factors (HSFs; Morimoto, 1998). The C. intestinalis harbors a single gene for HSF (Ci-Hsf; Imai et al., 2004). Upon heat shock, HSFs bind to sequences called heatshock elements (HSEs) and induce various genes involved in the heat-shock response. HSEs consist of three repeats of the nGAAn motifs and are classified into three types (Sakurai and Enoki, 2010): the perfecttype (nTTCn2GAAn2TTCn), the gap-type (nTTCn2GAAn7GAAn), and the step-type (nTTCn7TTCn7TTCn). Three HSEs were detected 247 to 169 bp upstream of the translation initiation site of Ci-HSPA1/6/7-like (Fig. 5a,c). The distal and proximal HSEs are perfect-type and gap-type, respectively. The intermediate HSE is step-type and shares one nGAAn motif with the distal HSE. The transcription start site determined by 50 -rapid amplification of cDNA ends (RACE) located 116 bp upstream of the translation initiation site (Fig. 5a,c). To test whether these HSEs were required for heatshock induction of Ci-HSPA1/6/7-like, we examined the activity of mutated plasmids. First, we conducted experiments using plasmids carrying successive 50 deletions of the upstream region of Ci-HSPA1/6/7-like. The eggs were electroporated with plasmid DNAs, allowed to develop up to the middle tailbud III stage, heat shocked at 28 C for 1 h, and examined for bgalactosidase activity by X-gal staining. The plasmids carrying the upstream region longer than 260 bp led to X-gal staining in >80% of electroporated embryos (Fig. 5b). The spatial pattern of X-gal staining in these embryos was essentially identical to that of embryos electroporated with plasmids that contained a 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7like and heat shocked at the middle tailbud III stage (Fig. 5b). No X-gal staining was observed with the plasmids carrying the 160 bp fragment (Fig. 5b). These

FIG. 3. Heat-shock response of LacZ expression in embryos electroporated with the plasmid construct that contains the 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like and LacZ. (a) Frequency of LacZ expression in electroporated embryos. The eggs were electroporated with the plasmid construct; allowed to develop up to the 32-cell, middle gastrula, middle neurula, early tailbud II, middle tailbud II, and middle tailbud III stages; heat shocked at 28 C for 1 h; and examined for b-galactosidase activity by X-gal staining. Control embryos were reared at 18 C. (b) Frequency of LacZ expression in each of tissues of electroporated embryos. The tissue was judged to be positive for LacZ expression if more than one cell showed X-gal staining. Note that the frequency of LacZ expression in tissues other than the muscle gradually decreased during embryogenesis. (c) Spatial expression patterns of LacZ detected by X-gal staining. Blue color in the embryos indicates that LacZ is expressed in that cell. Number of embryos examined is shown at the lower right. Bars indicate the boundary between the trunk and tail. epi: Epidermis, end; endoderm, mus; muscle, mes; mesenchyme, not; notochord.

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FIG. 4. Comparison of expression patterns of LacZ under the control of different regulatory elements. The eggs were electroporated with plasmid construct that contains the 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like, 367 bp fragment of the 50 -upstream region of Ci-Hsp24.4, or 2,600 bp fragment of the 50 -upstream region of Ci-EF1a; allowed to develop up to the middle tailbud III stage; heat shocked at 28 C for 1 h; and examined for b-galactosidase activity by X-gal staining. The number of embryos examined is shown at the lower or higher right corner. The staining were mainly detected in the muscle of embryos that were introduced with the plasmid construct that contains the 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like or one that contains the 367 bp fragment of the 50 upstream region of Ci-Hsp24.4. In contrast, embryos introduced with the plasmid construct that contains the 2,600 bp fragment of the 50 upstream region of Ci-EF1a ubiquitously exhibited the staining irrespective of whether they were heat shocked.

results suggest that the essential elements that mediate the heat-shock induction of Ci-HSPA1/6/7-like exist between 260 bp and 160 bp upstream of the translation initiation site. These results also suggest that 260 to 2,000 bp fragments of the 50 -upstream region of CiHSPA1/6/7-like can be similarly used for heat-inducible gene expression. Following this, we examined the activity of the plasmids with a deletion in the region containing HSEs. A deletion of all the three HSEs almost completely eliminated X-gal staining in the electroporated embryos (Fig. 5d). A deletion of the intermediate and proximal HSEs reduced the rate of embryos exhibiting X-gal staining to 21%. Neither a deletion of the distal and intermediate HSEs nor a deletion of the proximal HSE decreased the rate of embryos exhibiting X-gal staining. These results suggest that heat-shock induction of CiHSPA1/6/7-like depends on the presence of HSEs and that some complementarity exists among HSEs. Therefore, it is likely that heat-shock induction of Ci-HSPA1/ 6/7-like is controlled by Ci-Hsf.

Effects of Heat-Shock Treatments in Different Conditions on Normality of Embryos and Induction of Transgene Expression A possible concern regarding the application of a heat-inducible gene expression technique to C. intestinalis embryos is that embryos may be damaged by heatshock treatment. To determine the embryonic damages caused by heat-shock treatment, we examined the morphology of the embryos after heat shock. The cleavage stage embryos were heat shocked at 23 C or 28 C for 30 min or 1 h, cultured up to the larva stage, and examined for larval morphology. In control embryos reared at 18 C, 35% of the embryos showed normal morphology, 23% of the embryos had moderate defects such as bent tail, short tail, and/or deformed trunk, and the rest of the embryos showed more severe abnormalities (Fig. 6a–d). The ratio of normal embryos decreased when embryos were heat shocked at 23 C for 1 h from the 32-cell or 64-cell stage; at 28 C for 30 min from the 16-cell, 32-cell, or 64-cell stage; or at 28 C for 1 h from the 8-cell, 16-cell, or 32-cell stage (Fig. 6a). Heat-shock

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treatment at 28 C for 1 h from the 8-cell or 16-cell stage resulted in a particularly low rate of normal embryos and thus seemed unfit for analysis of gene function. We next examined the efficacy of a range of heatshock conditions in the induction of transgene expression. The fertilized eggs were electroporated with the plasmid DNA carrying the 2,000 bp fragment of the 50 upstream region of Ci-HSPA1/6/7-like and the LacZ reporter, allowed to develop up to the middle gastrula stage, heat shocked under six different conditions, and examined for b-galactosidase activity by X-gal staining (Fig. 7a). Heat-shock treatment at 28 C for 1 h resulted in broad X-gal staining in approximately 97% of embryos as expected (Fig. 7a,b). In contrast, when embryos were treated at 28 C for 30 min and then at 18 C for 30 min, the percentage of embryos positive for X-gal staining was approximately 16% and the staining was restricted to small regions of the embryo (Fig. 7a,c). Treatment at 28 C for 15 min and then at 18 C for 45 min reduced the rate of embryos showing X-gal staining to approximately 1.5% (Fig. 7a). Furthermore, the staining was limited to a few cells (Fig. 7d). Heat shock treatment at 23 C for 1 h, 30 min, or 15 min induced no X-gal staining at all (Fig. 7a). Rate of normal embryos appeared to be unaffected by heat-shock treatment under the six conditions applied (Fig. 7a). Application of the Heat-Inducible Gene Expression Technique to Analysis of Gene Function Finally, we verified the usefulness of the heatinducible gene expression technique through overexpression experiments on the well-characterized gene Ci-Bra (Corbo et al., 1997). This gene encodes a T-box transcription factor and plays an essential role in notochord formation. During normal development, fibroblast growth factor (FGF) signaling that takes place around the 32-cell stage induces Ci-Bra expression in the notochord precursor cells by the 64-cell stage (Yasuo and Hudson, 2006). When embryos are treated with the MEK inhibitor U0126, neither Ci-Bra expression nor notochord formation occurs (Yasuo and Hudson, 2006). We generated the plasmid construct that contains the 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like and Ci-Bra cDNA. The fertilized eggs were electroporated with this construct and cultured with U0126. When 32-cell stage embryos were heat shocked at 28 C for 1 h, reared up to the middle neurula stage at 18 C, and examined for the expression of a notochord marker Ci-fibrn (Hotta et al., 2000), 41% of the embryos expressed Ci-fibrn (Fig. 8a,b). When heat-shock treatment was continued up to the middle neurula stage, 55% of the embryos expressed Ci-fibrn (Fig. 8a,b). Without heat-shock treatment, 6% of the embryos expressed Ci-fibrn (Fig. 8a,b). No Ci-fibrn expression was detected in nonelec-

troporated control embryos (Fig. 8a,b). These results suggest that Ci-Bra induced by heat shock rescues a defect in the notochord formation in embryos deficient in FGF signaling and the gene expression technique using the cis-regulatory region of Ci-HSPA1/ 6/7-like is useful for inferring gene function in C. intestinalis embryos. CONCLUSION In this study, we developed a heat-inducible gene expression technique that exploits the heat-shock response of Ci-HSPA1/6/7-like, which enables conditional gene expression in C. intestinalis embryos. There are several issues that one should consider when using the present technique. First, one must optimize the temperature, duration, and timing of heat shocktreatment by trial and error. These factors would depend on the gene of interest and the objective of the experiment and should be determined to induce sufficient expression of the gene of interest, minimizing embryo damage. Based on the results of our experiments, the recommended condition of heat-shock treatment for a first trial is treatment at 28 C for 1 h after the 32-cell stage. Second, the results of the present study suggest that there is leaky expression from the construct carrying the cis-regulatory region of Ci-HSPA1/6/ 7-like under normal conditions. Such leakiness was observed in both assays with LacZ reporter constructs and analysis of Ci-Bra function. This can be explained by a characteristic of Ci-HSPA1/6/7-like, which shows weak basal expression under normal conditions. Therefore, care should be taken to avoid a possible problem caused by such leaky expression. The third issue is related to the result that heat shock after the neurula stage led to reporter gene expression in limited regions of the embryo. The result suggests that if one aims to induce a gene of interest after the neurula stage by the present technique, it is necessary to confirm whether the gene of interest is expressed in the desired regions. The present technique may be more useful when combined with other technologies. For example, in combination with methods of laser-mediated induction of heat-shock response that were developed and used in several animals (Deguchi et al., 2009; Kamei et al., 2009), the present technique helps in localizing the induction of gene expression. Furthermore, in combination with genome engineering technologies that were shown to be available in C. intestinalis (Kawai et al., 2012; Treen et al., 2014), the present technique offers strategies for not only overexpression experiments but also conditional gene targeting experiments. Altogether the present technique provides a useful tool for studies on the developmental biology of C. intestinalis.

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METHODS Biological Materials C. intestinalis cultivated at the Maizuru Fisheries Research Station of Kyoto University, Kyoto, and the Misaki Marine Biological Station of the University of Tokyo, Kanagawa, Japan, was used in the present study. The preparation of eggs and sperms, dechorionation of fertilized eggs, and culture of dechorionated embryos were performed as described previously (Yamada et al., 2003). Developmental stages of embryos were determined according to Hotta et al. (2007). For MEK inhibitor treatment, embryos were treated with 2 mM U0126 (Calbiochem). Control embryos without U0126 were treated with 0.1% dimethylsulfoxide. Heat-Shock Treatment Embryos were cultured on an agar-coated plate with Millipore-filtered seawater containing 50 mg/mL streptomycin (MFSW) at 18 C up to the desired developmental stage. The normal embryos were collected under a stereomicroscope and transferred with a minimal volume of MFSW to a new agar-coated plate with MFSW prewarmed at the specified temperature. During the operation, embryos were kept at 18 C. The plate was incubated at the specified temperature. Control embryos were incubated at 18 C using the same method. A set of heat-shock experiments was conducted using embryos from the same batch. Electroporation and X-Gal Staining Electroporation and X-gal staining were conducted as described previously (Oda-Ishii and Di Gregorio, 2007). Whole Mount In Situ Hybridization Whole mount in situ hybridization and preparation of digoxigenin-labeled probes were performed as described previously (Satou et al., 2001). The templates for the synthesis of probes were prepared from the following cDNA clones: cilv072a23 for Ci-HSPA1/6/7-like and XABT155657 for Ci-fibrn. The negative controls were prepared using probes for GFP.

Plasmid Construction All plasmids were produced using the multicassete Gateway vector set (Roure et al., 2007) and PCR Cloning System with Gateway Technology (Invitrogen) according to the manufacturer’s instructions. To create entry clones carrying the cis-regulatory region of a given gene, the 50 -upstream region of the gene was amplified by two rounds of polymerase chain reaction (PCR), and BP reactions were performed between the PCR products and a donor vector (pDONR-221-P3-P5; Roure et al., 2007). For the entry clone that contains the cis-regulatory region of Ci-HSPA1/6/7-like (pENTRL3-Ci-HSPA167like US 2K-L5), genomic DNA that corresponds to the 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like was amplified using primers for the first round of PCR (50 -ATAAAGTAGGCTAT ATTGGTAAGGTCCTTCAAAAAC-30 and 50 -CAAAAGTTG GGTTCTTGTAATCTTCTGAATATC-30 ) and those for the second round of PCR (attB3 adapter, 50 -GGGGACAAG TTTGTATAATAAAGTAGGCT-30 and attB5 adapter, 50 GGGGACCACTTTGTATACAAAAGTTGGGT-30 ). For the entry clone that contains the cis-regulatory region of CiHsp24.4 (pENTR-L3-Ci-Hsp24.4 US 367-L5), genomic DNA that corresponds to the 367 bp fragment of the 50 upstream region of Ci-Hsp24.4 was amplified using primers for the first round of PCR (50 -ATAAAGTAGGCTTT TTAATAATTCGCCTTAACTTATC-30 and 50 -CAAAAGTTG GGTTTCGCTTTTTTAGAATATTTCTTCA-30 ) and those for the second round of PCR (attB3 adapter and attB5 adapter). For the entry clone that contains the cis-regulatory region of Ci-EF1a (pENTR-L3-Ci-EF1a US 2.6KL5), genomic DNA that corresponds to the 2,600 bp fragment of the 50 -upstream region of Ci-EF1a was amplified using primers for the first round of PCR (50 -ATAAAGTAGGCTGTGTCCATGAAGGTAAGCGCTAT AG-30 and 50 -CAAAAGTTGGGTTTTGGAAGGTTGGGGT TAACCGA-30 ) and those for the second round of PCR (attB3 adapter and attB5 adapter). To create the expression clone that contains the cis-regulatory region of Ci-HSPA1/6/7-like and the LacZ reporter (pSP1.72-B3-Ci-HSPA167like US 2K-B5::B1-NLSlacZB2), the cis-regulatory region of Ci-Hsp24.4 and the LacZ reporter (pSP1.72-B3-Ci-Hsp24.4 US 367-B5::B1-

FIG. 5. Characterization of the cis-regulatory region of Ci-HSPA1/6/7-like. (a) The genomic structure of Ci-HSPA1/6/7-like. The open reading frame of Ci-HSPA1/6/7-like is shown in light blue. The 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like examined in the present study is indicated by a line. (b) Diagrams showing the 50 -upstream regions of Ci-HSPA1/6/7-like used for mutant plasmids carrying successive 50 -deletions. The frequency of embryos that showed X-gal staining in response to the heat-shock treatment and the number of embryos examined are shown on the right side. The inset indicates expression pattern of LacZ in embryos electroporated with plasmid carrying the 260 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like. (c) Nucleotide sequence of the 500 bp upstream region of the translation initiation site and the initiation codon of Ci-HSPA1/6/7-like. The nGAAn motifs in the perfect-type, gap-type, and step-type HSEs are shaded in yellow, magenta, and green, respectively. The middle nGAAn motif of the perfect-type HSE is identical to the distal nGAAn motif of the gap-type HSE. The transcription start site determined by 50 -RACE is colored red. The initiation codons of CiHSPA1/6/7-like and the upstream putative gene KH.L46.14 are colored light blue and orange, respectively. (d) Diagrams showing the 50 upstream regions of Ci-HSPA1/6/7-like used for mutant plasmids with a deletion in the region containing HSEs. The frequency of embryos that showed X-gal staining in response to the heat-shock treatment and the number of embryos examined are shown on the right side. The nGAAn motifs in the perfect-type, gap-type, and step-type HSEs are indicated by yellow, magenta, and green boxes, respectively.

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FIG. 6. Effect of a range of heat-shock treatments on the morphology of C. intestinalis embryos. (a) Frequency of morphological abnormalities in heat shocked embryos. The cleavage stage embryos were heat shocked at 23 C or 28 C for 30 min or 1 h, cultured up to the larva stage, and examined for larval morphology. Control embryos were reared at 18 C. Embryos were classified into normal embryos, embryos with incomplete neural tube closure, or embryos with severe defects according to their morphology. The number of embryos examined is shown above the graph. (b–d) Morphology of heat shocked embryos. Embryos with severe defects (b), with moderate defects (c), and with normal morphology (d) are shown.

NLSlacZ-B2), or the cis-regulatory region of Ci-EF1a and the LacZ reporter (pSP1.72-B3-Ci-EF1a US 2.6K-B5::B1-NLSlacZ-B2), LR reactions were performed between the previously described entry clones and a destination vector (pSP1.72BSSPE-R3-ccdB/ cmR-R5::B1-NLSlacZ-B2; Roure et al., 2007). To create mutated plasmids carrying successive 50 -deletions of the upstream region of Ci-HSPA1/6/7-like, entry clones carrying the 1,500-, 1,000-, 500-, 360-, 260-, or 160 bp (relative to the translation initiation site) fragments of the 50 -upstream region of Ci-HSPA1/6/7-like were constructed (pENTR-L3-Ci-HSPA167like US 1.5KL5, pENTR-L3-Ci-HSPA167like US 1K-L5, pENTR-L3-CiHSPA167like US 500-L5, pENTR-L3-Ci-HSPA167like US 360-L5, pENTR-L3-Ci-HSPA167like US 260-L5 and pENTR-L3-Ci-HSPA167like US 160-L5). Each fragment was amplified by two rounds of PCR, and BP reactions were performed between the PCR products and a donor vector (pDONR-221-P3-P5). Primers used for

the first round of PCR were as follows: 167US1.5K_B3, 5 0 -ATAAAGTAGGCTACTTGATTAACAAAGTATGAG-3 0 ; 167US1K_B3, 5 0 -ATAAAGTAGGCTTCAATGTATTGTT TACAATTTAG-30 ; 167US500_B3, 50 -ATAAAGTAGGCTAG TTTGCAGTCGTTCGTCGT-30 ; 167US360_B3, 50 -ATAAA GTAGGCTTATGTTTGTATTGTATTATG-30 ; 167US260_B3, 50 -ATAAAGTAGGCTATTGAATCGTCGGGAAACTTC-30 ; 167US160_B3, 50 -ATAAAGTAGGCTTTTATCCTCGTG CGTATATAATG-30 ; and 167DS_B5, 50 -CAAAAGTTGG GTTCTTGTAATCTTCTGAATATC-30 . Primers used for the second round of PCR were the attB3 adapter and the attB5 adapter. The mutated entry clones were used for LR reactions with the destination vector (pSP1.72BSSPE-R3-ccdB/cmR-R5::B1-NLSlacZ-B2) to create the expression clones (pSP1.72-B3-Ci-HSPA167like US 1.5K-B5::B1-NLSlacZ-B2, pSP1.72-B3-Ci-HSPA167like US 1K-B5::B1-NLSlacZ-B2, pSP1.72-B3-Ci-HSPA167like US 500-B5::B1-NLSlacZ-B2, pSP1.72-B3-Ci-HSPA167like US 360-B5::B1-NLSlacZ-B2, pSP1.72-B3-Ci-HSPA167like US

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FIG. 7. Effect of a range of heat-shock treatments on the expression of transgene. (a) Diagrams showing conditions of heat-shock treatments, frequency of LacZ expression in embryos, and frequency of normal embryos. The eggs were electroporated with the plasmid DNA carrying the 2,000 bp fragment of the 50 -upstream region of Ci-HSPA1/6/7-like and the LacZ reporter, allowed to develop up to the middle gastrula stage, heat shocked under six different conditions, and examined for b-galactosidase activity by X-gal staining and morphology. (b) Spatial expression patterns of LacZ detected by X-gal staining in embryos heat shocked at 28 C for 1 h. (c) Spatial expression patterns of LacZ detected by X-gal staining in embryos heat shocked at 28 C for 30 min and then at 18 C for 30 min. (d) Spatial expression patterns of LacZ detected by X-gal staining in embryos heat shocked at 28 C for 15 min and then at 18 C for 45 min.

260-B5::B1-NLSlacZ-B2, and pSP1.72-B3-Ci-HSPA167like US 160-B5::B1-NLSlacZ-B2). To create mutated plasmids with a deletion in the region containing HSEs, a deletion was introduced to pENTR-L3-Ci-HSPA167like US 2K-L5 using the KOD Plus Mutagenesis Kit (Toyobo) according to the manufacturer’s instructions. Primer pairs used for PCR were as follows: 167USDEL247_UR, 50 -CCGACGAT TCAATCAATATCAG-30 ; 167USDEL232_UR, 50 -GCTTCGC GAAGTTTCCCGAC-30 ; 167USDEL187_UR, 50 -GCGTTGA CGCGCGCCACCTA-30 ; 167USDEL_240DF, 50 -GCGAAGC TTCGCGCAATTTC-30 ; 167USDEL_220DF, 50 -GTGTTCTG AAAATAGGTGGC-30 ; and 167USDEL_170DF, 50 -GGT 167USDEL247_UR and TCTCCATTTATCCTCGT-30 . 167USDEL_240DF were used to create pENTR-L3-CiHSPA167like US 2KD247-240-L5. 167USDEL247_UR and 167USDEL_220DF were used to create pENTR-L3-CiHSPA167like US 2KD247-220-L5. 167USDEL247_UR and 167USDEL_170DF were used to create pENTR-L3-CiHSPA167like US 2KD247-170-L5. 167USDEL232_UR and 167USDEL_220DF were used to create pENTR-L3-CiHSPA167like US 2KD232-220-L5. 167USDEL232_UR and 167USDEL_170DF were used to create pENTR-L3-CiHSPA167like US 2KD232-170-L5. 167USDEL187_UR and 167USDEL_170DF were used to create pENTR-L3-CiHSPA167like US 2KD187-170-L5. The mutated entry clones were used for LR reactions with the destination vector (pSP1.72BSSPE-R3-ccdB/cmR-R5::B1-NLSlacZ-B2)

to create the expression clones (pSP1.72-B3-Ci-HSPA167like US 2KD247-240-B5::B1-NLSlacZ-B2, pSP1.72-B3-CiHSPA167like US 2KD247-220-B5::B1-NLSlacZ-B2, pSP1. 72-B3-Ci-HSPA167like US 2KD247-170-B5::B1-NLSlacZB2, pSP1.72-B3-Ci-HSPA167like US 2KD232-220-B5::B1NLSlacZ-B2, pSP1.72-B3-Ci-HSPA167like US 2KD232-170B5::B1-NLSlacZ-B2, and pSP1.72-B3-Ci-HSPA167like US 2KD187-170-B5::B1-NLSlacZ-B2). To create entry clones carrying the cDNA of Ci-Bra, the open reading frame of the gene was amplified by two rounds of PCR using primers for the first round of PCR (50 -AAAAAGCAGGCTCAGAAAAAATGACGTCATCAGATAGTAAGTT-30 and 50 -AGA AAGCTGGGTTTACAAAGAAGGTGGCGTAACGG-30 ) and those for the second round of PCR (attB1 adapter, 50 GGGGACAAGTTTGTACAAAAAAGCAGGCT-30 and attB2 adapter, 50 -GGGGACCACTTTGTACAAGAAAGCTGGGT-30 ). Following this, BP reactions were performed between the PCR products and a donor vector (pDONR-221) to create the entry clones (pENTR-L1-Kozak-Ci-Bra-stop-L2). To create the destination vector that contains the cis-regulatory region of Ci-HSPA1/6/7-like and the RfA cassete (pSP1.72BSSPE-B3-Ci-HSPA167like US 2K-B5::RfA), a BP reaction was performed between the fragments of pDONR221 corresponding to the ccdB/CmR region and pSP1.72-B3-Ci-HSPA167like US 2K-B5::B1-NLSlacZ-B2. To create the expression clone that contains the cis-regulatory region of Ci-HSPA1/6/7-like and Ci-Bra cDNA (pSP1.72-

HEAT-INDUCIBLE GENE EXPRESSION IN CIONA

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FIG. 8. Application of the heat-shock inducible gene expression technique to analysis of gene function in C. intestinalis embryos. (a) Frequency of Ci-fibrn expression in embryos. The eggs were electroporated with the plasmid construct that contains the cis-regulatory region of Ci-HSPA1/6/7-like and Ci-Bra cDNA, cultured with the MEK inhibitor U0126, heat shocked at the 32-cell stage, and examined for the expression of the notochord marker Ci-fibrn at the middle neurula stage. The heat-shock treatment for 1 h as well as up to the middle neurula stage (for 3.8 h) of embryos electroporated with the plasmid construct rescued Ci-fibrn expression that is otherwise inhibited by U0126. (b) Spatial expression patterns of Ci-fibrn visualized by whole mount in situ hybridization. The number of embryos examined is shown at the lower right.

B3-Ci-HSPA167like US 2K-B5::B1-Ci-Bra-B2), an LR reaction was performed between pSP1. 72BSSPE-B3-Ci-HSPA167like US 2K-B5::RfA and pENTR-L1-Kozak-Ci-Bra-stop-L2. Plasmids used in this study will be provided upon request. 50 -RACE 50 -RACE was performed using the GeneRacer Kit (Invitrogen) according to the manufacturer’s instructions. Total RNA was prepared from approximately 500 heat-shocked juveniles as described previously (Fujiwara et al., 2010). Approximately 3.5 mg of total RNA was used for 50 -RACE. The gene-specific primer used for

the first PCR was as follows: 50 -GAGGTATGCTTCCGC AGTGTCCTTCA-30 . Nested PCR was performed using the following nested gene-specific primer: 50 -CACGTC GCCTTGATATTCCGCTTGAAG-30 . Nested PCR amplified a single PCR product of approximately 450 bp, which was cloned and sequenced.

ACKNOWLEDGMENTS C. intestinalis was provided by Kyoto University and University of Tokyo through the National Bio-Resource Project of the MEXT, Japan.

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