Comparison of <i>Coup-TF's</i> upstream and 5′UTR regions between <i>P.lividus</i> and <i>S.purpuratus</i>.

<p><b><i>A</i></b>: The 5′UTR and the upstream sequence of the PlCoup-TF gene, numbered from the −1930 position. The data were obtained by subcloning and sequencing a 2.5 kb fragment of the λ clone “<i>Φ</i>” insert (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109274#pone-0109274-g002" target="_blank">Fig. 2</a>). The shaded areas correspond to various degrees of homology with the corresponding sequence of <i>SpCoup-TF</i> as revealed by comparisons using the program BLAST. The lighter the shade, the lesser the homology is between the ortholog genes of the two species. Thus, the darkest shade corresponds to the 5′UTR sequence that exhibits the highest homology. The small black arrow denotes the transcription initiation site. Underlined is the upstream sequence of module <i>a</i>, which is not homologous between the two species. <b>B</b>: Graphic comparison of the 5′UTR and 5′ upstream sequences of the two orthologous genes, <i>PlCoup-TF</i> (top) and <i>SpCoup-TF</i> (bottom) using the Family Relations program (32). Crossbars joining the two sequences indicate homology, the thickest of which corresponds to the 5′UTR region.</p

Spatial expression patterns of site-specific mutations into module <i>a</i>.

<p><b>A</b>: Graphic presentation of the wt and mutant sequences of the three elements within module <i>a</i>. The top line depicts the 320 bp region of the wt module <i>a</i>, and the sequences that correspond to the sites RE1 (−453), RE2 (−432) and RE3 (−377) and their respective position. Each additional line shows the nucleotides that substitute for the wt sequence at each site. The bottom line depicts the double mutation, which deletes also the intervening sequences between RE1 and RE2. <b>B</b>: Composite pictures of embryos expressing GFP resulting from injection of wt and mutant module <i>a</i>. The wt module <i>a</i> and mutation −453 show GFP expression specifically in the ciliary band, while mutation −432 shows expression in ciliary band, endoderm and aboral ectoderm. Mutation −377 shows GFP expression in aboral ectoderm and the double mutation −453/−432 in aboral ectoderm, endoderm and ciliary band. All embryos were photographed at pluteus stage.</p

Embryonic cell lineage specific expression of the GFP reference gene for each of the upstream deletions, individual upstream segments and mutations.

<p>Cb: Ciliary band; Cb+: Ciliary band and other cell types; En+: Endoderm and other cell types; Ae+: Aboral ectoderm and other cell types; Me+: Mesenchyme and other cell types.</p><p>Embryonic cell lineage specific expression of the GFP reference gene for each of the upstream deletions, individual upstream segments and mutations.</p

Spatial expression patterns generated by the individual segments a–f fused to the GFP cassette.

<p><b>A</b>: Graphical positioning of the upstream <i>PlCoup-TF</i> segments (a–f) within the 1932 bp upstream sequence, which were individually fused to the <i>EpGFPII</i> reference gene. The numbers surrounding each black bar correspond to the nucleotide borders of each segment. Other designations are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109274#pone-0109274-g004" target="_blank">Figure 4A</a>. <b>B</b>: Composite pictures of embryos resulting from injection of segments a–c. Segment a results in GFP expression specifically in the ciliary band, while segments b and c show GFP expression in the ciliary band, endoderm and mesenchyme cells. All embryos were photographed at pluteus stage. Embryos injected with segments d, e and f did not exhibit GFP expression.</p

Specific binding of embryonic nuclear proteins to elements RE1, RE2 and RE3.

<p>The DNA binding specificity of transcription factors to the elements RE1, RE2 and RE3 was determined by electrophoretic mobility shift assays. NE: Nuclear Extract; SC: Specific Competitor. (−) and (+) denote omission and addition of nuclear extract or specific competitor to each reaction respectively. The arrows point to protein: DNA complexes, which are not formed in the presence of specific competitor.</p

Spatial expression pattern of the <i>PlCoup-TF</i> gene.

<p>In situ hybridization of <i>P.lividus</i> embryos with antisense and sense digoxygenin labeled PlCoup-TF probes. <b>a–f</b>: antisense; <b>g–l</b>: sense probe hybridization. The maternal PlCoup-TF mRNA seems evenly distributed in the egg and at the 16-cell stage embryo. Zygotic transcripts are expressed in the presumptive oral ectoderm at blastula and gastrula stages and in the ciliary band at prism and pluteus stages. E: Unfertilized egg; 16c: 16-cell stage embryo; B: Hatching blastula; G: Gastrula; P: Prism and Pl: Pluteus.</p

Identification of <i>PlCoup-TF</i> transcription initiation sites.

<p><b>A</b>: Electrophoretic analysis of the 5'-RACE products. The two arrows point to the major DNA bands produced by the nested PCR (lane 1). The 100 bp ladder (NEB) was used as DNA length reference (lane M). <b>B</b>: Sequence of the proximal PlCoup-TF promoter and the positions of the multiple transcription initiation sites (capital and lower case bold characters). The most upstream site was designated as +1. A putative CCAAT box is underlined at position −65. The translation initiation site is located at +544.</p

Spatial expression patterns generated internal deletions D1–D4 into module <i>a</i>.

<p><b>A</b>: Map of the internal deletions. Black boxes correspond to the deleted regions D1–D4. The numbers surrounding each box mark the borders of each deletion. EpAc refers to <i>Endo16′s</i> promoter and <i>CyIIa's</i> kozak sequences as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109274#pone-0109274-g004" target="_blank">figure 4A</a>. The two parallel bars within the black box at the right end of the graph denote that the <i>GFP</i> gene is not depicted on scale. <b>B</b>: Composite pictures of embryos resulting from injection of the internal deletions D1–D4. The entire module <i>a</i> shows GFP expression specifically in the ciliary band, deletion D1 in ciliary band, endoderm and aboral ectoderm and D2 in aboral ectoderm. D3 shows expression in ciliary band and endoderm while D4 shows expression in mesenchyme cells. All embryos were photographed at pluteus stage.</p

Spatial expression patterns generated by the upstream deletions of the GFP cassette.

<p><b>A</b>: Map of <i>PlCoup-TF's</i> upstream sequence (1930 bp) fused to the <i>EpGFPII</i> reference gene. The bended arrow marks the transcription initiation site. EpAc refers to <i>Endo16′s</i> basal promoter and <i>CyIIa's</i> kozak sequence and ATG. The numbers above the map indicate the starting point of each upstream deletion. <b>B</b>: Composite pictures (GFP epi-fluorescence over bright field image) of embryos resulting from injection of the corresponding deletions −1639 to −232. Constructs −1639, −1398 and −781 show GFP expression in ciliary band and a few mesenchyme cells. Construct −532 shows GFP expression only in ciliary band and construct −232, in the aboral ectoderm. All embryos were photographed at pluteus stage. A picture of an embryo injected with the construct −19 is not shown, since these embryos never exhibited any GFP expression.</p

Spatial expression patterns generated by upstream deletions D-a1 to D-a5 of module <i>a</i>.

<p><b>A</b>: Graphic presentation of the upstream deletions into module a (−532 to −212). Horizontal bars underneath module <i>a</i>, represent the size of each deletion (D-a1 to D-a5) and the numbers above them the corresponding upstream border. The numbers at the right of each bar correspond to the size of each fragment tested. EpAc refers to <i>Endo16′s</i> promoter and <i>CyIIa's</i> kozak sequences as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109274#pone-0109274-g004" target="_blank">figure 4A</a>. The two parallel bars within the black box at the right end of the graph denote that the <i>GFP</i> gene is not depicted on scale. <b>B</b>: Composite pictures of embryos resulting from injection of upstream deletions into module <i>a</i>. The entire module <i>a</i> shows GFP expression specifically in the ciliary band, while deletions D-a1, D-a2 and D-a3 show expression both in ciliary band and endoderm. Deletions D-a4 and D-a5 show expression in endoderm and mesenchyme cells. D-a5 shows GFP expression in skeletogenic mesenchyme cells (see text for non-specific expression caused by random integration of the GFP cassette). All embryos were photographed at pluteus stage.</p