Role of SoxB binding sites in the regulation of <i>cyp26a1</i> expression in the anterior neural plate (ANP).

<p>(A) Representation of the cANE region in the zebrafish genome; 3 predicted SoxB binding sites are indicated; the green channel represents the intensity of the anti-Sox2 ChipSeq signal in this region according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150639#pone.0150639.ref034" target="_blank">34</a>]. (B) A logo representing the composite consensus binding site for SoxB/Oct factors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150639#pone.0150639.ref033" target="_blank">33</a>] is aligned with the predicted Sox binding site (Sox_BS1) overlapping Motif1 (cANE 45–59). The mutSox TT->CC mutation destroying the Sox-binding half-site is indicated. (C-F) egfp in situ hybridization representing enhancer activity of cANE ΔSoxBS2-3, where both Sox_BS2 and Sox_BS3 have been deleted (C), compared with intact cANE (D), at the 90% epiboly stage, and enhancer activity of Motif1 mutation mutSox (F) in 1–222 context compared with wild type 1–222 (E), at the 75% epiboly stage. Dorsal views; anterior is to the left. (G) RT-PCR relative quantification of total <i>egfp</i> expression stable transgenic embryos for constructs cANE (1–310), 81–310, 1–222 and 1–222 mutSox, as well as non transgenic embryos (Ctl); error bars represent SEM; units are arbitrary.</p

Characterization of cANE activity as an early neural plate specific enhancer.

<p>(A-I,A’-I’) Compared expression patterns of <i>cyp26a1</i> (A-I) and <i>egfp</i> driven by cANE (A’-I’) during early embryonic development. (J) Double in situ hybridization showing <i>barhl2</i> expression domain (blue) exactly filling the gap in the <i>cyp26a1</i> expression domain (red). (A,B,C,E,G,A’,B’,C’,E’,G’) are lateral views. (d,f,h,I,D’,f’,h’,I’) are animal pole views. White arrowheads: anterior neural plate. Black arrowheads: blastoderm marginal zone. Arrows in (H-J): gap in the anterior neural plate domain of <i>cyp26a1</i> expression. All stages are indicated in the pictures. (k,k’,l,l’) The effect of 100 nM retinoic acid (RA) treatment between 2,5 hpf and 8,5 hpf on on stable transgenic cANE_endo:::<i>egfp</i> (K-K’) and cANE::<i>egfp</i> (L-L’) expression. (M) EGFP fluorescence in a 12 hpf stable transgenic cANE:::<i>egfp</i> embryo. Lateral view with dorsal to the left. (N) Schematic representation of cANE and all three reported retinoic acid responsive elements (R1, R2, R3) identified previously. cANE is located from -504 bp to -195 bp relative to <i>cyp26a1</i> ATG codon. An: animal, Vg: vegetal; V: ventral; D: dorsal, A: anterior, P: posterior, L: left, R: right, CTRL: control embryo, RA: retinoic-acid treated embryo.</p

Conservation of vertebrate <i>cyp26a1</i> promoter region with zebrafish cANE.

<p>Alignment of 4 teleostean (tetraodon, fugu, stickleback and medaka) and mouse and human <i>cyp26a1</i> promoter regions orthologous to zebrafish cANE. The most conserved regions are outlined with red (predicted SoxB binding sites 2 and 3) or purple (conserved block1,2,3) boxes. The green (Motif1) and blue (predicted SoxB binding site 1) boxed sequences lie in the non-evolutionarily conserved region of zebrafish cANE. Numbers correspond to nucleotide coordinates within the 310 nt zebrafish cANE.</p

Detailed dissection of cANE by transient expression of deletion constructs in zebrafish embryos.

<p>(A) Schematic view of the constructs used for dissection of the cANE module. All constructs contain one single fragment (black) from the cANE module, placed immediately upstream of the <i>gata2</i> minimal promoter, except for construct cANE_endo, where the <i>gata2</i> promoter is replaced by the <i>cyp26a1</i> endogenous promoter. Numbers correspond to nucleotide coordinates within the 310 nt zebrafish cANE. The hatched block (Motif1) represents the 12-bp difference between constructs 39–310 and 50–310; the highly conserved blocks are indicated by checkered patterns. (B-K) Enhancer activity of the cANE deletions shown in (A), assayed by <i>egfp</i> in situ hybridization in transient or stable (labelled Tg()) transgenic embryos. All embryos are between stages 10.5 and 11 hpf, viewed from the animal pole, with anterior on the left.</p

Expression of <i>ISL1</i> and <i>GATA4</i> transcripts in the human heart between 26 and 38 days of gestation.

<p><b>A–H</b>: <i>ISL1 in situ</i> at Carnegie stages (CS)12 (26–28 days post fertilization [dpf]), CS13 (28–31 dpf), CS14 (32–33 dpf) and CS15 (34–36 dpf) respectively. <b>E–H</b> are magnifications of <b>A–D</b> respectively. <b>I–K</b> show <i>GATA4</i> expression in adjacent sections to <b>B–D</b>. <b>A</b>: <i>ISL1</i> is expressed at CS12 in foregut endoderm, splanchnic mesoderm, and early motoneurons. <b>B, F</b>: At CS13, <i>ISL1</i> is transcribed by mesenchyme around the cardiac OFT and pharyngeal arches. <i>ISL1</i> expression continues in the splanchnic mesoderm between the trachea and OFT, and is visible in dorsal root ganglia, at CS14 (<b>C, G</b>) and CS15 (<b>D, H</b>). <b>I–K</b>: <i>GATA4</i> is expressed in the endocardium and myocardium of the arterial pole at CS13, CS14 and CS15 (<b>I, J, K</b> respectively). <b>Inset</b>: RT-PCR of <i>ISL1</i>, <i>GATA4</i>, <i>GATA5</i>, <i>GATA6</i>, <i>FGF10</i> and positive control <i>ACTB</i> mRNAs in embryonic human hearts at stages CS13-16 (to 40 dpf). Abbreviations: drg, dorsal root ganglia; es, esophagus; fb, forebrain; fg, foregut; ph, pharynx; nt, neural tube; oft, OFT; ra, right atrium; t, trachea. Arrows, motoneurons. Bar: 110 µm (A–D, I) and 55 µm (E–H, J, K).</p

Bioinformatics analyses of the human <i>FGF10</i> locus surrounding the first exon.

<p><b>A</b>: Alignment of genomic regions around and within the human [hg18] <i>FGF10</i> locus to those of frog [xenTro2], chicken [galGal3], opossum [monDom4], mouse [mm9], dog [canFam2] and rhesus macaque [rheMac2] with colored regions >90% identical and the vertical scale ranging from 50% (bottom) to 100% (top). Color code for genomic features at <a href="http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html" target="_blank">http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html</a>. The <i>FGF10</i>-Pr1, <i>FGF10</i>-Pr2 and FGF10-Int1 regions examined in this study are boxed. <b>B</b>: A non-canonical predicted site for GATA-type transcription factors is 52 nucleotides 5′ to the ISL1 cognate sequence in <i>FGF10</i>-Int1 in the direction of transcription on the – strand in humans, mice and (not shown) macaque and opossum. <b>C</b>: Nucleotide sequence of the <i>FGF10</i>-Int1 enhancer module and position of conserved putative transcription factor binding sites as predicted by rVista (<a href="http://rvista.dcode.org" target="_blank">http://rvista.dcode.org</a>). All indicated human sites are identical to those of the macaque and mouse except for the SMAD prediction, only found in mouse; the ISL1, GATA and HOXA7 sites are also identical to the opossum, and the ISL1, NKX2-5 and TBX sites are also identical to the dog.</p

Sites of β-galactosidase activity in transgenic mouse embryos.

<p>All sites showed only selective cells positive for enhancer activation. DRGs = dorsal root ganglia; E = embryonic day of gestation; MN = motoneurons; OFT = cardiac outflow tract; PA = pharyngeal arch; PSM = pre-somitic mesoderm.</p

<i>In vitro</i> reporter assays support an additive combinatorial effect of transcription factors upon the FGF10 intronic enhancer.

<p>LUC-<i>FGF10</i>-Int1, which construct placed the luciferase gene under the control of the FGF10-Int1 element, was transfected alone or together with <i>ISL1</i>, <i>GATA4</i> and <i>TBX20</i> expression vectors into 10T1/2 cells. Each factor alone potentiated luciferase expression and these effects were additive in combination.</p