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

<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

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

Indomethacin treatment and survival of ctnnb1Δex3 mice.

<p>Indomethacin treatment results in the closure of WT and ctnnb1Δex3 ( = <i>Tyr::Cre/°; ctnnb1Δex3</i>/+) DA and allows the survival of ctnnb1Δex3 mice. Mock (A, B) and indomethacin (indo, 10 mg/kg body weight) (C, D) intraperitoneal injections into pregnant <i>Tyr::Cre/Tyr::Cre</i>; +/+; <i>Dct::LacZ/Dct::LacZ</i> females carrying <i>Tyr::Cre/</i>°; +/+; <i>Dct::LacZ/</i>° (A, C) and <i>Tyr::Cre/</i>°; <i>ctnnb1Δex3</i>/+; <i>Dct::LacZ/</i>° (B, D) E18.5 embryos. Four hours later, embryos were isolated, fixed, X-gal stained, transversally sectioned through the DA and counterstained with eosin. We treated three pregnant females and sectioned ten embryonic hearts (five WT and five mutants). The ductus arteriosus was closed in all cases. Note that the numbers of Dct+ cells derived from ctnnb1Δex3-Dct embryos obtained from pregnant mothers injected with indomethacin or mock-injected were similar. (E) Kaplan-Meier curves of WT and ctnnb1Δex3 newborn pups treated or mock-treated with indomethacin (6 mg/kg body weight indomethacin within 12 hours of birth). Ultrasound analysis was performed on treated versus non-treated animals during the second and third months, which associated survival of treated ctnnb1Δex3 to the size of the left atrium (not shown). Indomethacin-treated ctnnb1Δex3 mice survived significantly longer than mock-treated mice (p<0.009). Note similar results were obtained when ctnnb1Δex3 mi mice were treated with indomethacin or mock. Scale bars, (A, B)  = 100 µm, (C, D)  = 50 µm.</p

Histological analysis of WT and ctnnb1Δex3 lungs at P28.

<p>(A) WT ( = <i>Tyr::Cre</i>/°; +/+) and (B) ctnnb1Δex3 mice. Note the disorganized alveolae of the mutant lung. Nonetheless, the lung cells do not express the ctnnb1Δex3 transgene, suggesting that the effect is cell non-autonomous. Scale bars, (A, C, D)  = 50 µm, (B)  = 20 µm.</p

Melanoblasts are numerous in ctnnb1Δex3 DA.

<p>Ventral view of WT-Dct (A) and ctnnb1Δex3-Dct (B) E18.5 hearts stained with X-gal. Note that ctnnb1Δex3-Dct samples contain numerous β-galactosidase-stained cells (arrow) in the ductus arteriosus (DA). High magnification of the WT-Dct (C) and ctnnb1Δex3-Dct (D) DA regions, including the aorta (Ao) and the pulmonary trunk (PT). Scale bar (A, B)  = 1 mm.</p

Thrombosis develops in mutant mice during the second postnatal month.

<p>(A) Ultrasound analysis of a <i>Tyr::Cre</i>; <i>ctnnb1Δex3</i>/+ ( =  ctnnb1Δex3) mouse with dilated left atrium containing a thrombus. (B) Isolated thrombus. Expansion of the left atrium is observed prior to the appearance of thrombosis in such ctnnb1Δex3 mice: (C) at 6 weeks of age and (D) at 8 weeks, from the same animal. <i>In situ</i> thrombus located in a ctnnb1Δex3 left atrium prior to (E) and after dissection (F). Thr: (thrombus), LA: (left atrium), LV: (left ventricle) and Lu: (lung). Scale bar, B = 1 mm.</p

ctnnb1Δex3 mice die of heart failure between the second and fourth postnatal months.

<p>(A) Kaplan-Meier graph of survival of <i>Tyr::CreA/</i>°; <i>ctnnb1Δex3/</i>+ and WT littermate controls (<i>Tyr::CreA/</i>°; +/+ and °/°; <i>ctnnb1Δex3</i>/+). (B) Kaplan-Meier graph of survival of <i>Tyr::CreB</i>/°; <i>ctnnb1Δex3</i>/+ and WT littermate controls (<i>Tyr::CreB/</i>°; +/+). All members of both mutant populations perished before their fourth month of life, in contrast to the full survival of all their WT littermates.</p

Closure of the ligamentum arteriosum in ctnnb1Δex3 adult heart.

<p>The <i>Tyr::Cre; ctnnb1Δex3</i>/+ ( =  ctnnb1Δex3) ligamentum arteriosum (LigA) is not fully closed, rendering it a patent ductus arteriosus. At P28, the LigA does not usually show macroscopic hyperpigmentation in wildtype (WT) mice (arrows, A), whereas <i>Tyr::Cre; ctnnb1Δex3</i>/+ ( =  ctnnb1Δex3) LigA does (B). Transverse sections show that the WT LigA (C) is fully closed and does not contain any Mc, whereas ctnnb1Δex3 LigA (D–F) is only partially closed, containing both blood in the lumen and numerous pigmented melanocytes in the wall (E, F). The areas occupied by the intimal cushion (ic) and lumen (l) are shown in G and H, respectively. Cross-sections of the LigA show that the outer tunica is dense, while the inner ellipsoid part, known as the intimal cushion, has a distinct aspect. In ctnnb1Δex3 mice, a lumen is observable inside the ic. Ao: aorta, LigA: ligamentum arteriosum, Pa: pulmonary artery, Mc: melanocyte. Scale bars, (A, B)  = 0.25 mm, (C, D)  = 100 µm, (E)  = 50 µm and (F)  = 20 µm. For each genotype, the number of cells were estimated from 8-10 sections per LigA using 4 mice. *: p-value <0.05.</p