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).
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.
Download figure to PowerPoint
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.
Download figure to PowerPoint