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

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

Melanoblasts replace a proportion of the smooth muscle cells in the ctnnb1Δex3 DA.

<p>SMA-positive and X-gal-positive cells in transverse sections of E18.5 WT-Rosa and ctnnb1Δex3-Rosa DA were counted. Three categories of cells were distinguished: non-recombined SMC1 (SMA+ LacZ−), recombined SMC2 (SMA+ LacZ+), and recombined non-SMC (SMA− LacZ+), corresponding to melanoblasts (Mb). Note that the number of SMA+ LacZ+ SMC2 in WT-Rosa DA is similar to the number of SMA− LacZ+ Mb in ctnnb1Δex3-Rosa DA. Genotypes: WT-Rosa  =  <i>Tyr::Cre/</i>°; <i>+/+</i>; <i>Rosa26/</i>°, ctnnb1Δex3-Rosa  =  <i>Tyr::Cre/</i>°; <i>ctnnb1Δex3/+</i>; <i>Rosa26/</i>°. Note, the production of a mutated form of β-catenin in recombined cells did not seem to greatly affect the number of floxed cells, suggesting that there was no cell non-autonomous effect on the floxed SMC. In both panels, there were significant differences between the numbers of SMA+, LacZ+ cells and SMA−, LacZ+ cells (for each genotype, the number of cells were estimated from 5–8 sections per embryo using 4 embryos: ** p-value <0.01).</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

A Subpopulation of Smooth Muscle Cells, Derived from Melanocyte-Competent Precursors, Prevents Patent Ductus Arteriosus

<div><h3>Background</h3><p>Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The <em>ductus arteriosus</em> (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes.</p> </div

ctnnb1Δex3 is produced in melanoblasts and SMC cells of E18.5 DA.

<p>(A) The expression of β-catenin (<i>bcat</i>), <i>Mitf-M</i> (melanoblasts) and <i>Hprt</i> (loading control) was analyzed by RT-PCR on mRNA isolated from WT ( =  Tyr::Cre/°; +/+) and mut ( =  ctnnb1Δex3  =  Tyr::Cre/°; f3/+) DA at E18.5. M corresponds to the size marker. The “a” band (479 bp) corresponds to the non-recombined β-catenin cDNA or WT, whereas the “b” band (251 bp) corresponds to the recombined β-catenin cDNA or ctnnb1Δex3. <i>Ptgs2</i> (<i>Cox2</i>) is weakly expressed in WT and mutant DA. (B) Schematic of the WT versus mut <i>bcat</i> amplicons. (C–H) Immunolocalization of β-catenin in red (C, E, F, H), β-galactosidase in green (D, E, G, H) and DAPI in blue (E, H) in WT-Dct (C–E) and ctnnb1Δex3-Dct (F–H) sections of E18.5 DA. Note that β-catenin is found in both the cytoplasm and the nucleus of ctnnb1Δex3-Dct DA.</p

Abnormal circulation of the blood in ctnnb1Δex3 adult heart.

<p>(A) Ultrasound analysis showing an open <i>Tyr::Cre; ctnnb1Δex3</i>/+ (ctnnb1Δex3) DA. (B) Enlargement of (A) showing the DA. (C) Ultrasound analysis of a ctnnb1Δex3 patent foramen ovale. (D) Shunting through the foramen ovale as observed by echo Doppler analysis in ctnnb1Δex3 mice. (E) The foramen ovale remains open in ctnnb1Δex3 hearts. Note the presence of pigmented cells. LA: left atrium, RA: right atrium, Ao: aorta, RVOT: right ventricle outflow tract, DA: ductus arteriosus.</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

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