• ascidian;
  • chordate;
  • oligonucleotide microarray;
  • retinoic acid;
  • embryogenesis;
  • transcriptional regulation;
  • gene expression profile


  1. Top of page
  2. Abstract
  6. Acknowledgements

Oligonucleotide-based microarray analyses were carried out to identify retinoic acid target genes in embryos of the ascidian Ciona intestinalis. Of 21,938 spots, 50 (corresponding to 43 genes) showed over twofold up-regulation in retinoic acid-treated tail bud embryos. In situ hybridization verified retinoic acid-induced up-regulation of 23 genes. Many of them were expressed in the anterior tail region, where a retinaldehyde dehydrogenase homolog is expressed. Homologs of vertebrate genes involved in neurogenesis and/or neuronal functions (e.g., COUP-TF, Ci-Hox1, and SCO-spondin) were expressed in the central nervous system of Ciona embryos, and activated by retinoic acid. Genes encoding transcription factors (e.g., Ci-lmx1.2, vitamin D receptor, and Hox proteins) and apoptosis-related proteins (e.g., transglutaminase and an apoptosis-inducing factor homolog) were also activated by retinoic acid. Simultaneous treatment of embryos with retinoic acid and puromycin revealed a few direct targets, including genes encoding Ci-Hox1, Ci-Cyp26, and an Rnf126-like ring finger protein. Developmental Dynamics 233:1571–1578, 2005. © 2005 Wiley-Liss, Inc.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Retinoic acid (RA) is a vitamin A-derivative that regulates differentiation and morphogenesis of various embryonic and adult tissues in vertebrates (De Luca, 1991; Ross et al., 2000). Vitamin A-deficiency causes a variety of conditions such as the xerophthalmia, psoriasis, and growth inhibition (Wolbach and Howe, 1925). Embryos born to vitamin A-deficient females show developmental defects, including abnormalities in the hindbrain and the neural crest-derived craniofacial structures (Kalter and Warkany, 1959). Requirement of the RA signaling was also demonstrated by targeted disruption of genes encoding the RA-synthesizing aldehyde dehydrogenase (Raldh2; Niederreither et al., 1999) and RA receptors (RARs; Lohnes et al., 1994; Mendelsohn et al., 1994). Since orthologs of these genes were identified exclusively in chordates, acquisition of these genes was possibly a crucial event leading the chordate evolution (Nagatomo and Fujiwara, 2003; Fujiwara and Kawamura, 2003). In fact, many tissues whose development requires RA signaling (e.g., the dorsally located neural tube, neural crest cells, limbs, pharynx with the thyroid gland, and gill slits) are vertebrate-specific or chordate-specific (Fujiwara and Kawamura, 2003).

The genome of the protochordate ascidian Ciona intestinalis is as compact as that of Drosophila melanogaster and contains half the number of genes compared with that in the human genome (Dehal et al., 2002). However, the expression pattern of many developmental regulatory genes in ascidian embryos is similar to that in vertebrate embryos (Di Gregorio and Levine, 1998; Wada and Satoh, 2001). Therefore, Ciona provides a simple experimental system to study the evolutionary origin of chordate-specific gene regulatory networks such as RA signaling (Satoh et al., 2003).

The RAR is a DNA-binding transcription factor that activates gene expression upon binding RA (Mangelsdorf and Evans, 1995). Expression and function of the Ciona RAR gene (Ci-RAR) were analyzed (Nagatomo et al., 2003), and its target genes were identified by using a cDNA microarray containing 9,287 cDNA clones (Ishibashi et al., 2003). In the present study, many novel target genes were further identified, by using an oligonucleotide microarray containing 21,938 spots. They encoded candidate proteins responsible for RA-dependent cell proliferation and differentiation. In addition, we tested direct up-regulation of these target genes, using embryos that were treated simultaneously with RA and the translational inhibitor puromycin. The list of genes obtained here provides clues to understand the mechanism and evolutionary origin of vertebrate development and RA-related pathogenesis.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Identification of RA Target Genes by Oligonucleotide-Based Microarray Analysis

We previously performed a cDNA microarray analysis to identify RA target genes in the protochordate ascidian Ciona intestinalis (Ishibashi et al., 2003). To obtain further information, we carried out large-scale oligonucleotide microarray analysis. Ciona 22K custom oligo DNA microarrays were manufactured by Agilent Technologies. Each of 21,938 spots on a single glass slide contained unique 60-mer oligonucleotides derived from sequences of 17,834 independent cDNA clusters obtained from C. intestinalis cDNA project (Satou et al., 2002). This microarray covers approximately 85% of the transcripts (estimated to be around 16,000) of protein-coding genes in this species (Dehal et al., 2002).

Ciona embryos were continuously treated with 1 μM RA from the 32-cell stage to the initial tail bud stage. Gene expression patterns were examined using the oligo DNA microarray (Fig. 1A). We calculated the ratio of the fluorescent intensities resulting from an RA-treated probe and dimethyl sulfoxide (DMSO) -treated control probe (R:D). Reproducibility was confirmed by two hybridization experiments with swapped dyes. The average Cy5:Cy3 ratio of 162 deposition control spots within each slide was 0.1 ± 0.04 (experiment 1) and 0.08 ± 0.02 (experiment 2). When two or more spots correspond to a single gene, they showed a similar R:D ratio, suggesting the fidelity of experimental procedures (Table 1). To confirm the fidelity of our protocol, we also synthesized Cy5- and Cy3-labeled probes using the same RNA sample extracted from C. intestinalis fertilized eggs. The number of spots that showed the Cy5:Cy3 ratio greater than 2.0 or smaller than 0.5 was 14 (0.06%). As for spots showing absolute fluorescent intensity not less than 500, all showed the Cy5:Cy3 ratio in between 0.5 and 2.0. The coefficient of correlation for this experiment was 0.98. Thus, in this study, we recognized those showing R:D greater than 2.0 as candidate RA target genes.

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Figure 1. Scatter plot analysis comparing levels of expression in retinoic acid (RA) -treated and control (dimethyl sulfoxide [DMSO] -treated) embryos. For each experiment, individual spots were plotted using the log of the normalized signal intensity for Cy3 and Cy5 probes. The thick line is the linear regression line. Thin lines indicate the cutoffs (±2.0 fold). A: Comparison of the expression pattern obtained from RA-treated and control initial tail bud embryos. B: Gene expression in gastrulae treated with RA and puromycin, with reference to that obtained from treatment with DMSO and puromycin. C: Gene expression in gastrulae treated with RA, compared with that in DMSO-treated embryos. The amplitude of RA-induced activation in the presence of puromycin was smaller than that in the absence of puromycin, suggesting that puromycin inhibited indirect activation of gene expression by RA treatment.

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Table 1. Genes That Were Upregulated in the Ciona intestinalis Embryo by RAa
Cluster IDPutative protein product (sequence similarities)R:D (oligochip)R:D (cDNA)PuromycinIn situ
  • a

    Gene ID is as assigned in the C. intestinalis EST project database ( Putative protein products are essentially according to the annotation in the C. intestinalis genome databases ( R:D values (cDNA) were obtained in the previous study (Ishibashi et al., 2003). R:D values for puromycin-treated gastrulae (puromycin plus) are the geometrical average calculated from data obtained from two experiments. R:D values for control gastrulae (puromycin minues) are obtained from a single experiment. Underlines show spots whose R:D value was not less than 1.5 in control gastrulae (puromycin minus). Upregulation and/or ectopic expression confirmed by in situ hybridization were indicated (+ or ±). NA, Complementary DNA clones were not available; ND, not detected by in situ hybridization.

  • The most similar protein names were assigned to genes that have not yet been annotated.

00968rlNuclear receptor COUP-TF8.316.411.
34769rlEukaryotic translation initiation factor 3a (eIF3a p170)10.911.010.9-1.01.7NA
34769rlEukaryotic translation initiation factor 3a (eIF3a p170)4.715.48.5-0.91.2NA
32695rlNo similarity7.78.68.1-1.01.1NA
01311rlNo similarity6.
32306rlDopa/tyrosine sulfotransferase4.59.36.5-1.31.0+
06668rlHomo sapiens protein BAP285.
02210rlCi-Cyp26 (cytochrome P450 RA degrading enzyme)
16745rlNo similarity5.66.15.9-1.11.2+
06668rlHomo sapiens protein BAP283.
16745rlNo similarity4.36.25.1-1.00.9+
10360rlNADPH-flavin reductase4.
04977rlMus musculus Rnf126 ring finger protein3.
14025rlCi-lmx1.2 (LIM homeodomain protein)
03301rlCiona intestinalis serine protease-like4.33.74.0-1.11.4+
15755rlSimilar to C. elegans glp-
13490rlNo similarity3.
03301rlCiona intestinalis serine protease-like3.23.73.4-1.11.4+
34769rlEukaryotic translation initiation factor 3a (eIF3a p170)
00840rlSolute carrier family 6 (neurotransmitter transporter)
03348rlVitamin D receptor3.
13225rlDynein heavy chain4.
03348rlVitamin D receptor2.
00848rlNo similarity2.
04817rlInwardly rectifying potassium channel2.
31754rlVitamin D receptor2.33.82.9-1.21.1NA
02553rlHomo sapiens hypothetical protein XP_1748263.02.82.9(1.8, 1.4)0.60.9+
16905rlRetinol dehydrogenase2.72.82.8-1.21.0ND
35501rlCadherin-related 23 (Cdh23)
13982rlCi-Cyp26 (cytochrome P450 RA degrading enzyme)
03377rlp53-responsive apoptosis-inducing factor3.
00840rlSolute carrier family 6 (neurotransmitter transporter)
35486rlInwardly rectifying potassium channel3.32.22.7-0.90.9ND
01412rlOikopleura dioica similar to transmembrane receptor2.
34269rlSolute carrier family 22 (organic cation transporter)
03046rlLysosomal protective protein2.
15567rlNo similarity2.03.42.6-1.51.5ND
00124rlDrosophila melanogaster CG13902 protein2.
16687rlNo similarity2.13.02.5-0.80.8ND
02502rlSolute carrier family 1 (Glutamate transporter)
11854rlCytochrome P450 (CYP2 subfamily)±
12060rlNeural activity-related ring finger protein TRIM22.
30137rlParathyroid hormone receptor2.22.62.4-0.70.7ND
10761rlGonadotropin-releasing hormone II (GnRH-II)
36055rlNo similarity2.22.02.1-1.01.0NA

A geometrical average of R:D was calculated for each spot, based on two experiments. The top 72 spots marked the average R:D greater than 2.0. Fifty spots (corresponding to 43 genes) showed the R:D greater than 2.0 in both experiments (Table 1). In the previous study, up-regulation of 15 genes in RA-treated embryos was demonstrated by cDNA microarray and in situ hybridization (Ishibashi et al., 2003). Among them, 14 were also in the oligo DNA microarray used in this study, and 12 exhibited an average R:D greater than 2.0. Table 1 contains 11 of them (ID 00840r1, 00968r1, 01311r1, 01890r1, 02210r1, 02553r1, 03348r1, 04817r1, 04977r1, 10360r1, and 13982r1). In general, average R:D values were higher in the present study, probably due to low background signal characteristic to oligochips.

Identification of Direct RA Target Genes

It appeared that many genes were regulated by transcription factors whose expression was directly activated by RAR. We tried to distinguish direct RA targets by simultaneous treatment of embryos with 1 μM RA and 200 μg/ml puromycin. For reference, embryos were treated with 0.1% DMSO and 200 μg/ml puromycin. Because puromycin affects cell division and further morphogenesis by inhibiting protein synthesis, we treated embryos for only 1 hr from the 64-cell stage to avoid detecting toxic side effects of puromycin on gene expression patterns. Hybridization experiments were carried out twice, and the geometrical average of the R:D ratio was calculated (Table 1). As a control experiment for puromycin treatment, embryos were treated with 1 μM RA for an hour from the 64-cell stage in the absence of puromycin. For reference, embryos were treated with 0.1% DMSO. After a 1-hr treatment at 20°C, puromycin-treated embryos looked like the 64-cell embryo, whereas control embryos developed into the early∼middle gastrula (data not shown). In this experiment, the magnitude of up- and down-regulations was relatively small probably because of the short period of drug treatment (Fig. 1B,C).

Genes that showed high R:D values irrespective of the presence or absence of puromycin were candidate direct targets. In contrast, those exhibiting a high R:D value only in the absence of puromycin were thought to be secondary targets. Of 50 spots shown in Table 1, 10 (underlined) exhibited an R:D value greater than 1.5 in the absence of puromycin. Among them, six spots showed similar or greater R:D values in the presence of puromycin (Table 1). These included the genes encoding putative RA degrading enzyme Ci-Cyp26 (02210r1 and 13982r1), Ci-Hox1 (01890r1), a ring finger-containing protein similar to mouse Rnf126 (04977r1), solute carrier family 22 organic cation transporter (34269r1). The first two were well-known direct RA target genes in vertebrates (White et al., 1997; Manzanares et al., 2000).

Table 2 shows 18 spots whose R:D value was greater than 1.8 in the presence of puromycin. Most of them exhibited similar R:D values irrespective of the presence of puromycin (Table 2). These included two spots corresponding to an iodothyronine deiodinase homolog (00141r1), and two spots corresponding to a gene encoding a WD repeat-containing protein (34642r1). Except for 02210r1 (Ci-Cyp26) and 04977r1 (similar to mouse Rnf126 ring finger protein), R:D values in the tail bud-stage embryo were around 1.0 (Table 2). This finding suggests that many direct RA target genes are transiently activated after RA treatment. We prepared lacZ reporter constructs containing genomic fragments corresponding to 02210r1, 01890r1, and 04977r1. These reporter genes were expressed in the Ciona embryo and strongly activated by RA (data not shown). A search for RA response elements (RARE) in these genomic DNA fragments is currently under way.

Table 2. Genes That Were Upregulated by RA in the Presence of Puromycin
IDPutative protein product (sequence similarities)PuromycinR:D (oligo)R:D (cDNA)
02210rlCi-Cyp26 (cytochrome P450 RA degrading enzyme)
04977rlMus musculus Rnf126 ring finger protein of unknown function2.
00141rlIodothyronine deiodinase2.
00141rlIodothyronine deiodinase2.
10598rlAnkyrin repeat and SAM domain containing protein2.
00095rlSimilar to Ciona intestinalis hypothetical protein (Q95P26)
37039rlNo similarity2.
31817rlDNA repair protein Rad502.
05750rlScavenger receptor cysteine-rich protein1.
34642rlWD repeat-containing proteins1.
31789rlEarly endosomal protein1.
12450rlChromosome-associated kinesin KIF41.
12317rlGap junction α4 protein (connexin 37)
31874rlMitotic kinesin-like protein1.
32727rlZinc finger-containing protein of unknown function1.
00435rlCreatine kinase1.
34642rlWD repeat-containing proteins1.

Spatial Expression Pattern of RA Target Genes

In the previous study, we observed RA-induced up-regulation of 00840r1, 00968r1, 01311r1, 01890r1, 02210r1, 02553r1, 03348r1, 04817r1, 04977r1, 10360r1, and 13982r1 by in situ hybridization (Ishibashi et al., 2003). Among them 10 showed high fluorescent intensity (over 1,000) in this study (data not shown). In contrast, most of the newly listed genes showed fluorescent intensity lower than 1,000. These results suggest that, in the previous study, we detected most of RA target genes whose amount of mRNA was abundant in the embryo. In this study, in situ hybridization revealed up-regulation and/or ectopic activation of 00124r1, 00897r1, 01121r1, 02502r1, 03301r1, 03377r1, 06668r1, 13225r1, 13988r1, 14025r1, 16745r1, and 32306r1 in RA-treated embryos (Table 1; Fig. 2). For most other genes, expression was not detected in either RA-treated or control embryos, probably because of low amounts of mRNAs in the embryo.

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Figure 2. Spatial expression pattern of retinoic acid (RA) target genes in RA-treated and dimethyl sulfoxide (DMSO)-treated control embryos. A–K: Expression of 04977r1 (A), 01890r1 (B), 02210r1 (C), 13988r1 (D), 00124r1 (E), 14205r1 (F), 04817r1 (G), 02553r1 (H), 01311r1 (I), 03301r1 (J), and 32306r1 (K). Panels d1–d3 show DMSO-treated control embryos, and r1–r3 show RA-treated embryos. Panels d1 and r1 show gastrulae, d2 and r2 show initial tail bud embryos, and d3 and r3 show early–middle tail bud embryos. The anterior is oriented to the left. Panels d1 and r1 show a vegetal (dorsal) view. Panels d2, d3, r2, and r3 show a lateral view, except that panel d2 of A and J, r2 of A, G, H, I, and J show a dorsal view. Arrows indicate the staining in the anterior tail epidermis. C,D,F,H: Arrowheads indicate the staining in the central nervous system (C,D,F) or the adhesive organ containing sensory neurons (H). H,I,K: Red arrows show ectopic staining in the epidermis (H,I) or endoderm (K). Red arrowheads show the presumptive brain cells in which RA-induced ectopic activation was not observed.

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Figure 2 shows the spatial expression pattern of selected RA target genes in RA-treated and control (DMSO-treated) embryos. In control embryos, many RA target genes were expressed in the anterior tail region (Fig. 2A–C,G–J). Particularly, candidate direct target genes (04977r1, 01890r1, and 02210r1) were expressed in the epidermis and nerve cord of this region (Fig. 2A–C). ID 00968r1 (COUP-TF) was also expressed in the nerve cord of this region (Imai et al., 2004). These genes may be directly activated by endogenous RA, because a homolog of Raldh2 RA-synthesizing enzyme is expressed in the anterior-most tail muscle cells (Nagatomo and Fujiwara, 2003). Expression of 01890r1, 02210r1, 13988r1 (SCO-spondin), 00124r1 (similar to Drosophila melanogaster CG13902), 14025r1 (Ci-lmx1.2) was observed in the central nervous system of control embryos (Fig. 2B–F). COUP-TF is a transcription factor that is known to be involved in neurogenesis (Park et al., 2003). Mouse SCO-spondin is expressed predominantly in the brain and is thought to be involved in axonal pathfinding (Goncalves-Mendes et al., 2003). Transcription of a few lmx genes were affected by RA treatment in the Xenopus embryo (Arima et al., 2005). In RA-treated embryos, some genes were ectopically expressed in the epidermal lineage but excluded from the neural plate (Fig. 2H,I). Some other genes were activated by RA in the entire ectodermal tissues, including the central nervous system (Fig. 2A–C,G). This and the previous studies suggest that gene expression in the epidermis and central nervous system is sensitive to RA treatment (Ishibashi et al., 2003). Many symptoms related to vitamin A deficiency in vertebrates have been observed in the epidermis and central nervous system, suggesting that one of the most primitive and fundamental roles of RA in the chordates is to regulate ectodermal differentiation. Expression of 03301r1 (serine protease) was observed in a few epidermal cells in the posterior head region (Fig. 2J). Ectopic activation of this gene was induced by RA in the anterior head region (Fig. 2J). Expression of 32306r1 (dopa/tyrosine sulfotransferase) was not detected in control embryos (Fig. 2K). A small subset of endodermal cells expressed this gene in RA-treated embryos (Fig. 2K). Mammalian dopa/tyrosine sulfotransferase is expressed in the liver and is involved in the regulation of hormone activity and excretion of xenobiotics (Glatt et al., 2001). Expression of 00897r1 (Caveolin-1 homolog) and 02502r1 (solute carrier family 1 glutamate transporter) was observed in the tail muscle cells in control embryos, and activated by RA in all cell types (data not shown). ID 16745r1, 13225r1, 01121r1, and 03377r1 were ubiquitously expressed and up-regulated by RA (data not shown).

Characterization of RA Target Genes

As described above, many RA targets were expressed in the central nervous system (Fig. 2) and seem to be involved in neurogenesis and/or neuronal functions. They included transcription factors, such as COUP-TF (00968r1), Ci-Hox1 (01890r1), Ci-lmx1.2 (14025r1). Transcription factors comprised a major group of RA targets. Another transcription factor listed in Table 1 was a nuclear receptor Ci-VDR (03348r1 and 31754r1). Vertebrate orthologs of Ci-VDR encode the vitamin D3 receptor whose most important role is controlling mineral ion homeostasis (Christakos et al., 2003). 03348r1 was also listed in the previous cDNA microarray analysis, where it was annotated as a homolog of the orphan nuclear receptor LXR (Ishibashi et al., 2003).

Genes mediating the growth promoting activity of RA have not yet been characterized and, therefore, is important for further biological and clinical study. In the present study, three spots corresponding to a single gene (ID 34769r1) exhibited a high R:D ratio (Table 1). The R:D value was 1.0 in the presence of puromycin, whereas it was 1.6∼1.7 in the absence of puromycin (Table 1). This gene is a homolog of the largest subunit of eukaryotic translation initiation factor 3 (eIF3a; Browning et al., 2001). In vertebrates, overexpression of eIF3a resulted in down-regulation of the synthesis of the inhibitor of cyclin-dependent kinases, p27, suggesting that eIF3a regulates the cell cycle progression (Dong and Zhang, 2003). The g rowth-promoting activity of RA may be mediated in part by eIF3a, although vertebrate eIF3a is not reported to be up-regulated by RA.

A few apoptosis-related genes were activated by RA. They included 01121r1 (transglutaminase) and 03377r1 (p53-responsive apoptosis-inducing factor, AIF). Transglutaminase expression is activated by RA in a few human tumor cell lines during apoptosis (Piacentini et al., 1991). An RARE was identified in the upstream regulatory sequence of the mouse transglutaminase gene, demonstrating that transglutaminase gene is a direct RA target (Yan et al., 1996). AIF is involved in chromatin condensation and DNA fragmentation during apoptosis (Susin et al., 1999). Apoptosis-inducing pathway regulated by RA may have been established in the chordate ancestor.

We identified 23 RA target genes in the C. intestinalis embryo; among them at least six were direct RA targets. Based on these fundamental data, further functional analysis will be designed to reveal the RA-mediated gene regulatory cascade in the Ciona embryo. As stated in Introduction section, RA regulates development of various chordate-specific characteristics. Some are shared by all the chordate species and some are not. For example, neural crest cells and limbs have been established only in the vertebrate lineage of evolution. Therefore, comparison of the repertoire of target genes among different chordate taxa will provide clues to understand molecular mechanisms of the chordate evolution.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Biological Materials

Ciona intestinalis was obtained near the Usa Marine Biological Institute of Kochi University. Eggs and sperm were surgically obtained from the gonoducts. Embryos were treated with 1 μM all-trans RA (Sigma-Aldrich, St. Louis, MO) from the 32-cell stage to the initial tail bud stage. For reference, embryos were treated with 0.1% DMSO, since 1 mM RA stock was dissolved in DMSO. To examine direct response to RA, 1 μM all-trans RA treatment was carried out in the presence or absence of 200 μg/ml puromycin (Sigma-Aldrich) for 1 hr from the 64-cell stage. For reference, embryos were treated with 0.1% DMSO in the presence or absence of 200 μg/ml puromycin, respectively.

Microarray Analysis

A Ciona 22K custom oligo DNA microarray (Agilent Technologies, Palo Alto, CA) containing 21,938 oligonucleotides synthesized based on sequence data of the Ciona intestinalis cDNA project (Satoh et al., 2003) was used. Total RNA samples were purified from drug-treated and control embryos using the acid guanidine thiocyanate-phenol/chloroform (AGPC) method (Chomczynski and Sacchi, 1987), with slight modifications (Fujiwara et al., 1993). The quality of RNA was tested by electrophoresis using an Agilent 2100 bioanalyzer (Agilent Technologies). Total RNA (5 μg) was labeled with Cy3 or Cy5 using an Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Oligochips were hybridized with labeled probes. Hybridization and wash processes were carried out according to the manufacturer's protocol. The microarrays were scanned using a GenePix4000B DNA microarray scanner (Axon Instruments, Foster City, CA) and GenePix Pro 4.0 microarray analysis software (Axon Instruments) and GeneSpring software (Silicon Genetics, Redwood City, CA) were used for image analysis, normalization, and data extraction processes.

In Situ Hybridization

Spatial expression pattern was examined by in situ hybridization as described previously (Nagatomo et al., 2003).


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