Constitutive CAP1/Prss8 inactivation results in embryonic lethality.

<p>Distribution of genotypes following heterozygote breeding. Number of wildtype (WT, black) CAP1/Prss8 heterozygous mutant (Het, grey) and knockout (KO, white) mice at weaning (<b>A</b>). Survival curves in percentage of wildtype (black diamonds), heterozygous mutant (grey spares) and knockout (black diamonds) littermate embryos between E8.5 and E16.5. Note, that no knockout embryo survived beyond E14.5 (<b>B</b>). (<b>C</b>) Representative pictures of gross morphology of embryos at different developmental stages from wildtype (WT), heterozygous mutant (Het) and homozygous mutant (KO) mice. The black cross indicates dead/partially degraded embryos; ***<i>P</i><0.001.</p

CAP1/Prss8 knockout placentas present a late differentiation defect.

<p>Representative pictures of placenta. (<b>A</b>) Placental discs at E12.5 and E14.5. H&E stainings of sagittal sections of placentas at E12.5 and E14.5 from wildtype (WT), heterozygous (Het) and knockout (KO) embryos at (<b>B</b>) low (x50) and at (<b>C</b>) high magnification (x400). From day E12.5 onwards, maturation from cytotrophoblasts to syncytiotrophoblasts is observed in wildtype and heterozygous mutant but not in knockout. The dashed line indicates the limit between the labyrinth and the decidua in <b>B</b>, and surrounds the cytotrophoblasts in <b>C</b>.</p

CAP1/Prss8 knockout embryos present normal placentas.

<p>Epiblast-specific CAP1/Prss8 knockout (Lox/Δ cre+) pups were obtained by mating <i>CAP1/Prss8</i>^Δ<i>/+</i><i>;Sox2::Cre^Tg/+</i> to <i>CAP1/Prss8^lox/lox</i> mice. (<b>A</b>) Genotype distribution of embryos recovered at E14.5 and relative quantification of CAP1/Prss8 mRNA expression levels in placentas and embryos (<b>B</b>). <i>CAP1/Prss8^lox/</i>^Δ<i>;Sox2::Cre^Tg/+</i>, embryo-specific knockout (Lox/Δ Cre+), white bar; <i>CAP1/Prss8^lox/</i>^Δ (Lox/Δ Cre-) and <i>CAP1/Prss8^lox/+;Sox2::Cre^Tg/+</i>, heterozygous mutant (Lox/+ Cre+), grey bar; <i>CAP1/Prss8^lox/+</i> wildtype (Lox/+ Cre-); black bar. Note that the CAP1/Prss8 mRNA expression in the knockout (Lox/Δ Cre+) embryos is abolished while the corresponding control (Lox/+ Cre+) placentas show heterozygous expression levels. (<b>C</b>) H&E staining of sagittal placenta sections at E14.5 from controls (<i>CAP1/Prss8^lox/+</i>; Lox/+ cre-), heterozygotes (<i>CAP1/Prss8^lox/</i>^Δ; Lox/Δ) and embryo-specific knockouts (<i>CAP1/Prss8^lox/</i>^Δ<i>;Sox2::Cre^Tg/+</i>; Lox/Δ cre+).</p

Primers used for quantitative PCR.

<p>Primers used for quantitative PCR.</p

CAP1/Prss8 knockout embryos are born and exhibit an abnormal epidermis.

<p>Genotype distribution of pups at birth and at weaning (<i>CAP1/Prss8</i>^Δ<i>/+</i><i>;Sox2::Cre^Tg/+</i> x <i>CAP1/Prss8^lox/lox</i>). Two litters were taken right after birth (n = 21 pups, <b>A</b>) or following weaning (3 week-old; n = 26 pups, <b>B</b>) and genotyped. (<b>C</b>) H&E staining of sagittal backskin sections of one-day-old pups from controls (<i>CAP1/Prss8^lox/+</i>; Lox/+ cre-), heterozygous (<i>CAP1/Prss8^lox/</i>^Δ; Lox/Δ cre-) and embryo-specific knockouts (<i>CAP1/Prss8^lox/</i>^Δ<i>;Sox2::Cre^Tg/+</i>; Lox/Δ cre+). The latter displayed a compact stratum corneum (hyperkeratosis) indicative of a skin barrier defect. Four additional litters were genotyped at weaning; magnification x200.</p

mRNA transcription profile of placental-specific markers.

<p>Placental expression of CAP1/Prss8 (Prss8) (<b>A</b>) of wildtype (WT, black diamond), heterozygous (Het, grey box), and knockout (KO, open triangle) embryos. **Shows the statistical difference between wildtype and heterozygous level. (<b>B</b>) Overlay of the CAP1/Prss8 peak of expression in wildtype placenta (solid line) and the survival curve of knockout embryos (dotted line). (<b>C</b>) Relative mRNA expression levels of <i>Par2, pRb, Eomes and Gcm1</i> were determined in placenta from wildtype, heterozygous mutant and knockout embyros from E11.5 to E14.5, each time point representing the average value measured from at least 3 animals.</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

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

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