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Keywords:

  • ascidian;
  • enhancer;
  • Hox ;
  • retinoic acid;
  • transcriptional regulation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The Hox1 gene in the urochordate ascidian Ciona intestinalis (Ci-Hox1) is expressed in the nerve cord and epidermis. We identified a nerve cord enhancer in the second intron of Ci-Hox1, and demonstrated that retinoic acid (RA) plays a major role in activating this enhancer. The enhancer contained a putative retinoic acid-response element (RARE). Mutation of the RARE in the Ci-Hox1 nerve cord enhancer only partially abolished the enhancer activity. Genes encoding RA synthase and the RA receptor were knocked down using specific antisense morpholino oligos (MOs), and injection of embryos with these MOs resulted in the complete disappearance of epidermal expression of Ci-Hox1 and reduction of neural expression. However, nerve cord expression was not completely repressed. These results suggest that the nerve cord enhancer is activated by two partially redundant pathways; one RA-dependent and one RA-independent.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Hox genes play important roles in the establishment of segmental identities during embryogenesis in vertebrates (reviewed by Parrish et al. 2009). Inactivation of the Hoxa-1 gene in mice resulted in deletion or reduction of rhombomere 5 (r5) and severe reduction of r4 (Lufkin et al. 1991; Chisaka et al. 1992; Carpenter et al. 1993; Dollé et al. 1993; Mark et al. 1993), while patterning of r4 was lost in Hoxb-1 mutant mice (Studer et al. 1996). In zebrafish embryos, hoxb1a is required for migration of motor neurons in r4 (McClintock et al. 2002) and hoxb1b is required for patterning of r4 (McClintock et al. 2002). The expression of many Hox genes in vertebrates is regulated by retinoic acid (RA) (reviewed by Gavalas & Krumlauf 2000), and murine Hox genes include RA-response elements (RAREs) (Langston & Gudas 1992; Dupé et al. 1997; Langston et al. 1997; Huang et al. 1998, 2002). Excess RA and RA antagonist were shown to cause abnormal gene expression in the central nervous system in the cephalochordate amphioxus (Schubert et al. 2006). RA activates the expression of the amphioxus Hox1 gene (AmphiHox1), which in turn regulates the regionalization of the central nervous system (Schubert et al. 2004, 2006). AmphiHox1 contains a functional RARE in the 3′ flanking region (Manzanares et al. 2000; Wada et al. 2006).

Retinoic acid is necessary for morphogenesis in vertebrate embryos, as demonstrated by targeted disruption of RA receptors (RARs; Lohnes et al. 1994; Mendelsohn et al. 1994) and the retinaldehyde dehydrogenase Raldh2 (Niederreither et al. 1999). Notably, RA regulates the formation of chordate-specific organs and tissues, including the neural tube, branchial aches, paired limbs, and neural crest cells (reviewed by Fujiwara & Kawamura 2003; Fujiwara 2006; Marlétaz et al. 2006; Campo-Paysaa et al. 2008). RAR forms a heterodimer with retinoid X receptor (RXR), and binds to RAREs within the enhancer region of target genes (reviewed by Bastien & Rochette-Egly 2004).

The ascidian Ciona intestinalis is a marine invertebrate chordate. The phylum Chordata consists of three subphyla: urochordates (including ascidians), cephalochordates, and vertebrates. The tadpole-type larvae of ascidians represent the basic chordate body plan (reviewed by Satoh & Jeffery 1995; Di Gregorio & Levine 1998; Satoh et al. 2003). Ascidians show the closest evolutionary relationship to vertebrates (Putnam et al. 2008), and are thus key animals in understanding the evolution of vertebrates from invertebrates. The C. intestinalis genome contains a single Hox1 gene (Ci-Hox1) (Dehal et al. 2002; Ikuta et al. 2004), which is expressed in the visceral ganglion, nerve cord, and a restricted area of the epidermis (Nagatomo & Fujiwara 2003; Ikuta et al. 2004; Ikuta & Saiga 2007; Imai et al. 2009). Microarray analysis revealed that Ci-Hox1 expression was upregulated by RA in the absence of translation (Ishibashi et al. 2005), and RA was required for the epidermal expression of Ci-Hox1 (Kanda et al. 2009). This epidermal expression of Ci-Hox1 is required to form the atrial siphon primordia, homologous to the vertebrate otic placode (Sasakura et al. 2012). In a previous study, we identified and characterized a RARE in the 5′ flanking region of Ci-Hox1, which was responsible for its expression in the epidermis (Kanda et al. 2009). However, the regulatory elements driving Ci-Hox1 expression in the central nervous system have not yet been characterized.

In this study, we therefore further investigated the regulation of Ci-Hox1 expression in the central nervous system by characterization of a neural enhancer located in the second intron of Ci-Hox1.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals

Ciona intestinalis juvenile adults were kindly provided by Kazuko Hirayama, Chikako Imaizumi, Shota Chiba, Reiko Yoshida and Yutaka Satou at Kyoto University through the National Bio-Resource Project, MEXT, Japan. They were cultivated for several months in Tosa Bay near the Usa Marine Biological Institute of Kochi University. Gametes were obtained surgically from the gonoducts and the eggs were inseminated with non-self sperm. Fertilized eggs were dechorionated with 0.05% actinase E (Kaken Pharmaceutical) and 1% sodium mercaptoacetate (Wako Pure Chemical Industries). Embryos were raised in agarose-coated Petri dishes.

Transgenes

Genomic DNA corresponding to the 5′ flanking region of Ci-Hox1 was amplified by polymerase chain reaction (PCR) using the primers 5′-GTATTGCTGCAGACTAGTAGGTAAAG-3′ (in the 5′ flanking region) and 5′-ACCGGGTATTCTAGATACGAATTCATCTTACACCT-3′ (in exon 2). The PCR product was excised with PstI and XbaI. Genomic DNA corresponding to the second intron of Ci-Hox1 was amplified by PCR with the primers 5′-CACTCTCGAGTAACACTTACGATTG-3′ (in the 3′ end of exon 2) and 5′-GTTATCTGCAGTTACTCCGTAGGTG-3′ (in the 5′ end of exon 3). The PCR product was inserted into pGEM-T (Promega) to produce the plasmid pGEM-i2. The intronic fragment was then excised from pGEM-i2 with XhoI and PstI. Both fragments were inserted into the XhoI and XbaI sites of pSP72-1.27 (Corbo et al. 1997) to produce the plasmid Ci-Hox1(i2)/lacZ. Construction of Ci-Hox1(i2Δ1)/lacZ was achieved by PCR using the primers 5′-GGAAAGCTCGAGTACAAATCAAC-3′ (in intron 2) and 5′-GTTATCTGCAGTTACTCCGTAGGTG-3′ (in the 5′ end of exon 3). The PCR product was excised with XhoI and PstI and the fragment was inserted into the XhoI and PstI sites of Ci-Hox1(i2)/lacZ. Ci-Hox1(i2Δ2)/lacZ, Ci-Hox1(i2Δ3)/lacZ, and Ci-Hox1(i2Δ4)/lacZ were similarly constructed. The forward primers used for PCR were 5′-ATTGGCGCCTCGAGGGTTCGCCTC-3′, 5′-CACCAATACTCGAGCACCAAAAGG-3′ and 5′-CT-GCCCGACTCGAGCGGCTATGCG-3′, respectively (in intron 2), and the reverse primer was 5′-GTTATCTGCAGTTACTCCGTAGGTG-3′ (in the 5′ end of exon 3). For internal deletion to create Ci-Hox1(i2Δp2)/lacZ, PCR was performed using pGEM-i2 as a template, using the primers 5′-CGTTTTTCGACTTTCTGTTTATAAAATACATTAC-3′ (in intron 2) and 5′-CAAAAACAAATAGTGGACACATAGCCTTATTAGG-3′ (in intron 2). PCR was performed using KOD plus (Toyobo) to produce a blunt-ended product, which then underwent self-ligation catalyzed by T4 DNA ligase (Promega). The resultant plasmid, named pGEM-i2Δp2, contained a truncation within the intronic fragment. The truncated fragment was excised from pGEM-i2Δp2 with XhoI and PstI and the restriction fragment was inserted into the XhoI and PstI sites of Ci-Hox1(i2)/lacZ. Ci-Hox1(i2Δp3)/lacZ and Ci-Hox1(i2DR5mut)/lacZ were constructed using similar methods. PCR was performed using the following primers: 5′-CCGCTGCCCGACGCGAACGGCTATGCGTTAC-3′ (in intron 2) and 5′-CGGATAAAGGGACCATTGAAACGACAACGTTC-3′ (in intron 2) for Ci-Hox1(i2Δp3)/lacZ; and 5′-CATGAACTCTCCACTCTTTCGCTCACACTATATC-3′ (in intron 2) and 5′-AACCCAGCATCTACCCCGCGGCGTTTCGATA-TC-3′ (in intron 2) for Ci-Hox1(i2DR5mut)/lacZ.

Electroporation and drug administration

A large-scale preparation of plasmid DNA was carried out using QIAGEN tip-100 (Qiagen). The plasmid was dissolved in 500 μL of 0.77 mol/L mannitol at a concentration of 60 μg/mL. The plasmid was introduced into dechorionated embryos by electroporation, as described by Corbo et al. (1997). Embryos were treated with 1 μmol/L all-trans RA (Sigma-Aldrich), as described by Kanda et al. (2009).

Gene knockdowns

The custom-made antisense morpholino oligonucleotide (MO) sequences for Ci-Raldh2 and Ci-RAR were 5′-GTACTGCTGATACGACTGAAGACAT-3′ and 5′-CATAACGTTCATCATGCTATAGTAT-3′, respectively (Gene Tools; LLC). They were designed to target the sequence containing a putative initiation codon. The concentration of MO in the injection medium was 0.5 mmol/L, using 0.1 mg/mL Fast green (Wako) in distilled water as the injection medium. Unfertilized eggs were dechorionated as described above and injected with the MOs. After microinjection, the eggs were inseminated and allowed to develop in agarose-coated Petri dishes.

Whole-mount in situ hybridization

RNA probes were labeled with digoxigenin (DIG), as described by Nagatomo et al. (2003). Embryos were fixed with 4% paraformaldehyde in 0.1 mol/L 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.5) and 0.5 mol/L NaCl at 4°C overnight. They were rehydrated by stepwise transfer into 50% and 30% ethanol in phosphate-buffered saline containing 0.1% Tween 20 (PBST). The embryos were washed twice with PBST, treated with 2.5 μg/mL proteinase K in PBST at 37°C for 45 min, and then washed twice again with PBST. The embryos were post-fixed with 4% paraformaldehyde in PBST for 1 h, washed four times in PBST, then incubated in hybridization buffer (50% formamide, 5× standard saline citrate (SSC), 100 μg/mL tRNA, 50 μg/mL Denhardt's solution [Wako], 1% Tween20) at 42°C for more than 1 h. They were then incubated in hybridization buffer containing 0.5–1.0 μg/mL DIG-labeled probe. Hybridization was carried out at 42°C overnight. After hybridization, the specimens were washed three times in 4× wash buffer (50% formamide, 4× SSC, 0.1% Tween 20) at 50°C for 15 min, and twice in 2× wash buffer (50% formamide, 2× SSC, 0.1% Tween 20) at 50°C for 15 min. They were rinsed three times in solution A (0.5 mol/L NaCl, 10 mmol/L Tris–HCl (pH 8.0), 5 mmol/L ethylenediaminetetraacetic acid [EDTA], 0.1% Tween 20) at room temperature for 15 min, treated with 25 μg/mL RNase A in solution A at 37°C for 30 min, washed sequentially in solution A at room temperature for 15 min, 2× wash buffer at 50°C for 20 min, twice in 0.5× wash buffer (50% formamide, 0.5× SSC, 0.1% Tween 20) at 50°C for 15 min, and four times in PBST at room temperature for 15 min. The hybridization signal was detected immunologically, as described by Fujiwara et al. (2002). Images were taken using an AZ100 microscope (Nikon, Tokyo, Japan).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Characterization of the nerve cord enhancer of Ci-Hox1

The Ci-Hox1 gene consists of four exons. Our previous study suggested that the nerve cord enhancer of Ci-Hox1 was located within the second intron (Kanda et al. 2009). We therefore characterized this enhancer by constructing a reporter gene named Ci-Hox1(i2)/lacZ (Fig. 1A,B). The transgene contained the 160-bp 5′ flanking region, followed by the first exon, first intron, and first 22 bp of the second exon (Fig. 1A,B). The 160-bp 5′ flanking region had no enhancer activity (data not shown). The translated region of lacZ was connected downstream of a putative translation initiation site of Ci-Hox1 located in the second exon (Fig. 1B). The full-length (3.6 kb) second intron was fused upstream to the 160-bp 5′ flanking region (Fig. 1A,B). We have prepared a similar transgene, named Ci-Hox1(intron2)/lacZ, which contained only a 2-kb central region of intron2 (Kanda et al. 2009). Therefore, Ci-Hox1(i2)/lacZ differs from Ci-Hox1(intron2)/lacZ. The transgene was introduced into fertilized eggs by electroporation, and Ci-Hox1(i2)/lacZ was expressed in the nerve cord (Fig. 1C). Expression was restricted to the visceral ganglion and anterior nerve cord regions, in a pattern similar to that of endogenous Ci-Hox1 mRNA (Fig. 1C). Ci-Hox1(i2)/lacZ was activated by 1 μmol/L RA (Fig. 1D) and its expression was restricted to the dorsal side of the embryo (Fig. 1D,F,H,J). Neural tube closure is inhibited by RA treatment (Nagatomo et al. 2003), and most of the cells expressing Ci-Hox1(i2)/lacZ on the dorsal surface were thus thought to be prospective neural plate cells. The expression of lacZ in the cerebral vesicle was non-specific, because Ci-Hox1 was not expressed in this tissue (Fig. 1C,E,G,I), and this region of the cerebral vesicle often expresses reporter genes, even when they are not controlled by specific enhancer elements (Harafuji et al. 2002). The second intron sequence was gradually deleted from the 5′ side, and transgenes containing 3302, 2908, or 2330 bp of the intronic sequence produced similar expression patterns (Fig. 1E,G,I), and expression was activated by RA (Fig. 1F,H,J). The transgene containing 1828 bp of the intronic sequence (Ci-Hox1(i2Δ4)/lacZ) was not expressed in the nerve cord (Fig. 1K) and was not activated by RA (Fig. 1L). Some embryos expressed transgenes in mesenchyme cells (e.g. Fig. 1K,L), and this expression was also non-specific (Fig. 1K,L). The expression vector used in this study contained a sequence element able to activate lacZ transcription in mesenchyme cells (Robert W. Zeller, pers. comm., 2011). These results indicate that the 503-bp region (Element A) in the middle of the intronic sequence is important for neural expression and RA responsiveness of Ci-Hox1 (Fig. 1B, red line). The nucleotide sequence of Ci-Hox1 was compared with that of a homologous gene in the closely related species, C. savignyi, using the VISTA Genome Browser (http://genome.lbl.gov/vista/index.shtml) (Frazer et al. 2004). Four blocks of conserved sequences (peaks 1–4) were identified in the second intron (Fig. 2A). Peak 3 was located within Element A (Fig. 2A) and contained a RARE-like sequence (Fig. 2B). This sequence was highly conserved between the two Ciona species, and was similar to other functional RARE sequences (Fig. 2C,D).

image

Figure 1. Identification of the nerve cord enhancer of Ci-Hox1. (A) Genomic structure of the Ci-Hox1 gene. Exons are indicated by gray boxes. The putative transcription start site is indicated by an arrow. The putative translation start site is indicated by an arrowhead. (B) Diagrams showing the reporter constructs containing the second intron and the 5′ flanking region of Ci-Hox1. Names of transgenes are given at the upper right of each diagram. The length of the intronic fragment contained in the transgenes is shown under the diagrams. Recognition sites of restriction enzymes are indicated. sp, SpeI; ps, PstI. (C–L) Expression pattern of transgenes, visualized by in situ hybridization. In all panels, the anterior side of the embryo is oriented to the left, with the dorsal side up. nc, nerve cord; ns, non-specific expression. Names of transgenes are given at the bottom of each panel. (B, D, F, H, and J) dimethylsulfoxide (DMSO)-treated control embryos. (C, E, G, I, and K) retinoic acid (RA)-treated embryos.

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image

Figure 2. Conserved sequence elements in the second intron of the Hox1 gene in two related Ciona species. (A) Comparison of Hox1 orthologues between C. intestinalis and C. savignyi using the VISTA Genome Browser. Genomic structure is on the upper side of the waveform graph. The waveform represents the degree of sequence similarity and regions showing high similarity are colored: conserved regions within the coding sequence are blue and those within the non-coding sequence are red. Conserved regions within the second intron are numbered (peaks 1–4). (B) Nucleotide sequence of peak 3. A DR5-type retinoic acid-response element (RARE)-like sequence is indicated by a black box. The putative Ci-RAR-binding site is shaded in green and the putative Ci-RXR-binding site is in yellow. (C) Alignment of the nucleotide sequence of part of peak 3 between Ci-Hox1 and C. savignyi Hox1 (Cs-Hox1). The RARE-like sequence is boxed. (D) RARE sequences of the Hox genes. These data were compiled with reference to Mainguy et al. (2003), Wada et al. (2006), and Kanda et al. (2009). The RARE sequence identified in the current study is boxed. Hs, Homo sapiens; Mm, Mus musculus; Bf, Branchiostoma floridae; Ci, Ciona intestinalis.

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Neural expression of Ci-Hox1 is not completely dependent on RA

We generated an internal deletion of peak 3 of Element A in Ci-Hox1(i2)/lacZ, and the resultant reporter gene was named Ci-Hox1(i2Δp3)/lacZ (Fig. 3A). As described above, Ci-Hox1(i2)/lacZ was expressed in the nerve cord and activated by RA (Fig. 1C,D), while in contrast, expression of Ci-Hox1(i2Δp3)/lacZ in the nerve cord was weak (Fig. 3B) and was not activated by RA (Fig. 3C). A point mutation was introduced into the RARE-like sequence in Ci-Hox1(i2)/lacZ (Fig. 3L) to produce the plasmid Ci-Hox1(i2DR5mut)/lacZ (Fig. 3A). Nerve cord expression of Ci-Hox1(i2DR5mut)/lacZ was weak (Fig. 3D) and unresponsive to RA (Fig. 3E). We generated the transgene Ci-Hox1(i2Δp2)/lacZ as a control (Fig. 3A). Ci-Hox1(i2Δp2)/lacZ contained peak 3, but lacked peak 2 (Fig. 3A), exhibited neural expression in control embryos, and was activated by RA (Fig. 3F,G). We also constructed the reporter genes Ci-Hox1(i2Δ3-Δp3)/lacZ and Ci-Hox1(i2Δ3-DR5mut)/lacZ by introducing internal deletion and point mutations, respectively, in Ci-Hox1(i2Δ3)/lacZ (Fig. 3A). Ci-Hox1(i2Δ3)/lacZ was expressed in the nerve cord (Fig. 1I,J), while in contrast, Ci-Hox1(i2Δ3-Δp3)/lacZ and Ci-Hox1(i2Δ3-DR5mut)/lacZ were not expressed in the nerve cord (Fig. 3H,J) and did not respond to RA (Fig. 3I,K). These results suggest that the nerve cord enhancer in the second intron of Ci-Hox1 contains two distinct elements, including Element A, which contains a RARE, and activates Ci-Hox1 transcription in the nerve cord in control embryos in a RA-dependent manner. Exogenously administered RA can thus activate Element A. However, deletion or point mutation of the RARE within the full-length intron only partially suppressed the enhancer activity, suggesting the existence of another weak enhancer element located within the 5′ 1282-bp region of the second intron, which is present in Ci-Hox1(i2)/lacZ but not in Ci-Hox1(i2Δ3)/lacZ (Fig. 3A, blue bidirectional arrow).

image

Figure 3. The retinoic acid-response element (RARE)-like sequence in the second intron of Ci-Hox1 is necessary for responsiveness to RA. (A) Diagrams showing the reporter constructs containing internal deletions and point mutations within the conserved regions. Names of transgenes are given at the upper right of each diagram. Exons of Ci-Hox1 are indicated by gray boxes. The putative transcription start site is indicated by an arrow. The putative translation start site is indicated by an arrowhead. The mutated site is indicated by a red cross. (B–K) Expression pattern of transgenes visualized by in situ hybridization. In all panels, the anterior side of the embryo is oriented to the left, with the dorsal side up. nc, nerve cord; ns, non-specific expression. Names of transgenes are given at the bottom of each panel. (B, D, F, H and J) dimethylsulfoxide (DMSO)-treated control embryos. (C, E, G, I and K) RA-treated embryos. Expression pattern of Ci-Hox1(i2Δ3)/lacZ is shown in Figure 1I,J. (L) Comparison of the RARE-like sequence and its mutated form (RARE mut). The DR5-type RARE-like sequence is boxed. The putative Ci-RAR-binding site is shaded in green and the putative Ci-RXR-binding site is in yellow. Lower-case letters indicate mutated nucleotides.

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Disruption of the RA signal causes downregulation of Ci-Hox1

Genes encoding RA synthase (Ci-Raldh2) and Ci-RAR were knocked down using specific antisense MOs. Epidermal expression of Ci-Hox1 was completely abolished in embryos injected with either one of these MOs, and neural expression was severely affected (Fig. 4B,D). However, expression in the nerve cord was not completely repressed (Fig. 4B′,D′). These results suggest that epidermal expression of Ci-Hox1 was completely dependent on RA, while neural expression was activated by two partially redundant pathways: one RA-dependent and one RA-independent.

image

Figure 4. Disruption of Ci-Raldh2 and Ci-RAR by antisense morpholino oligos (MOs). All panels show the expression patterns of endogenous Ci-Hox1. (A) Tail-bud embryos developed from eggs injected with lacZ-specific control MO. (B) Tail-bud embryos carrying a Ci-Raldh2-specific MO. (C) Tail-bud embryos carrying a lacZ-specific control MO. (D) Tail-bud embryos carrying a Ci-RAR-specific MO. (B′ and D′) High magnification images of the nerve cord expressing Ci-Hox1. In all panels, the anterior side of the embryo is oriented to the left, with the dorsal side up. ep, epidermis; nc, nerve cord.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

RARE in the Ci-Hox1 second intron is necessary for RA-responsiveness in the nerve cord

The results of this study provide evidence to support a major role for RA in activating the nerve cord enhancer of Ci-Hox1. The enhancer is located in the second intron, which was shown to include a highly conserved DR5-type RARE. The point mutation in Element A rendered the enhancer completely insensitive to RA, suggesting that the RARE is necessary for RA-responsiveness in the nerve cord. In addition, the mutation severely impaired enhancer activity in control embryos, suggesting that RA is responsible for the transcriptional activation of Ci-Hox1 during embryogenesis. Natale et al. (2011) reported that the 3′ portion (1.7 kb) of the Ci-Hox1 second intron showed enhancer activity in the nerve cord. Because the RARE is located near the 5′ end of the 1.7-kb region, it is unclear if the RARE was contained in the 3′ portion; however, it is likely that the 1.7-kb fragment contained the RARE.

Ci-Hox1 gene has two RAREs

We previously found a DR2-type RARE in the 5′ flanking region, which regulated the epidermal expression of Ci-Hox1 (Kanda et al. 2009), but not the expression of reporter genes in the nerve cord (Kanda et al. 2009). The neural RARE identified in the current study, however, did not activate the epidermal expression, suggesting that these two RAREs have tissue-specific functions. Expression of the murine Hoxb-1 gene is regulated by three RAREs (reviewed by Marshall et al. 1996): one is located in the 5′ flanking region and represses the expression in r3/5 (Studer et al. 1994); the other two are located in the 3′ flanking region and activate the expression of Hoxb-1. One of these is required in the neural epithelium and the other is required in the foregut (Marshall et al. 1994; Huang et al. 1998, 2002). The human HOXB1 gene enhancer has two RAREs, accompanied by binding sites for different transcription factors (Ogura & Evans 1995a,b). In transgenic mice, one RARE was shown to be required for the establishment of early HOXB1 expression in the primitive streak-stage embryo, and the other was involved in the establishment of r4-specific expression (Ogura & Evans 1995b). In these cases, the RARE alone was not sufficient to activate transcription, suggesting that cooperation with other transcription factors is necessary for full enhancer activity and tissue specificity (Ogura & Evans 1995b). The involvement of tissue-specific transcription factors in the regulation of Ci-Hox1 is unclear, and further detailed studies are needed to reveal the mechanism of tissue-specific transcriptional regulation of Ci-Hox1.

Ci-Hox1 expression is also regulated by an RA-independent mechanism

Imai et al. (2009) demonstrated that a Ci-Raldh2-specific MO reduced the expression of Ci-Hox1. We also confirmed that the MO completely silenced the epidermal expression of Ci-Hox1, but the results of the present study showed that the MO did not completely suppress Ci-Hox1 expression in the nerve cord. Similarly, a Ci-RAR-specific MO completely suppressed the expression of Ci-Hox1 in the epidermis but not in the nerve cord. These observations are consistent with the results of disruption of the RARE in the second intron, which similarly failed to suppress the enhancer activity in the nerve cord completely. Imai et al. (2009) showed Ci-Hox1 mRNA in the initial tail-bud stage, while we examined expression in the middle tail-bud stage. Natale et al. (2011) revealed that the 5′ flanking region of Ci-Hox1 contained another nerve cord enhancer responsible for activation during the larval stage, and it is possible that the 5′ upstream enhancer was responsible for the late expression of the Ci-Hox1 mRNA observed in our MO-injected embryos. However, the weak activation of the intronic enhancer lacking the RARE cannot be explained, because the reporter construct did not contain the 5′ upstream enhancer. We therefore propose that the intronic nerve cord enhancer is activated by at least two different transcription factors: an RAR/RXR heterodimer that binds the RARE in Element A, and another, RA-independent transcription factor(s). Binding sites for the latter factor(s) seem to be located in the 5′ 1282-bp region of the second intron. Deletion of the 5′ region did not seriously affect enhancer activity, suggesting that the contribution of the 5′ element is much smaller than that of Element A. Further studies are required to identify the transcription factor(s) involved in the RA-independent transcriptional activation of Ci-Hox1 in the nerve cord.

Evolution of the Hox1 enhancer in chordates

The enhancer element located in the 3′ flanking region of AmphiHox1 is necessary and sufficient to activate the expression of reporter genes in both the neural tube and neural crest cells in chick embryos (Wada et al. 2006), and the RARE within this enhancer is essential for transcriptional activation (Wada et al. 2006). The cephalochordate amphioxus diverged from the other chordate groups, including urochordates and vertebrates, suggesting that the RA-dependent transcriptional activation of Hox1 should originate from the common chordate ancestor. The subphylum Urochordata consists of ascidians, salps, and larvaceans. The spatial expression pattern of the Hox1 gene in the larvacean Oikopleura dioica is similar to that of Ci-Hox1, and Oikopleura Hox1 is expressed in the epidermis and neural tube (Seo et al. 2004; Cañestro et al. 2005; Cañestro & Postlethwait 2007). However, the O. dioica genome does not contain genes encoding Raldh2, RAR, or the RA-degrading enzyme Cyp26 (Cañestro et al. 2006), and treatment with RA does not interfere with embryogenesis in O. dioica (Cañestro & Postlethwait 2007). In addition, exogenously administered RA did not activate Hox1 in O. dioica (Cañestro et al. 2006), and Hox1 expression was unaffected by a pharmacological inhibitor of RA synthesis (Cañestro & Postlethwait 2007). The results of the current study suggest the existence of an RA-independent pathway regulating the expression of Ci-Hox1. This RA-independent transcriptional machinery might originate from the common ancestor of urochordates and vertebrates.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This work was supported in part by a Research Fellowship for Young Scientists (#11J11025) and Grants-in-Aid from the Japan Society for the Promotion of Science (#22570214). We would like to thank the National Bioresource Project, MEXT, and all members of the Maizuru Fishery Research Station of Kyoto University. We are particularly grateful to the late Ms Kazuko Hirayama who helped us set up the culture system in Kochi University. We also thank Zenji Imoto at the Usa Marine Biological Institute of Kochi University for collecting animals.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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