Proliferation defects in conditional <i>p600</i> knockout heart.

<p>Transverse sections of <i>p600</i>^−/+ (<i>p600</i>^+/−/<i>Sox2-Cre</i>⁺) (A) and <i>p600</i> cKO (B) heart at days E13.5 were stained with anti-Ki-67 antibody. After immunehistological staining, the samples were counterstained with hematoxylin. Magnified images of the ventricular wall (top) and the interventricular septum (bottom), the region indicated by rectangles, are shown in the right panels. Scales bars in the left and right panels indicate 500 and 200 µm, respectively. (C) The percentage of Ki-67 negative cells in the ventricular wall and the interventricular septum are shown. The <i>p</i>-values for Student’s t-test are shown in the graph. ** and *** show <i>p</i>-value <0.01 and <0.001, respectively.</p

Disruption of <i>p600</i> results in growth retardation and lethality during embryonic development.

<p>(A) Targeting strategy of <i>p600</i> locus. The exon encoding the initiating methionine codon of <i>p600</i> allele was replaced with a Neo cassette by homologous recombination in ES cell lines. The ES cell lines were microinjected into mouse blastocysts to generate chimeric mice. These chimeras were bred to obtain offspring that are heterozygous for ‘Neo allele’. <i>p600</i>^Neo/+ mice were then crossed with <i>EIIa-Cre</i> transgenic mouse <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066269#pone.0066269-Lakso1" target="_blank">[19]</a> to delete the loxP flanked region, yielding ‘KO allele’. The locations of PCR amplicons used for genotyping are as indicated. Restriction enzyme digestion of genomic DNA with BamHI and SpeI produces the 2.5 and 4.3 kbp fragments from WT and Neo alleles, respectively, that hybridize with the probe for Southern blotting. The regions for PCR genotypings are indicated. (B) The genotypes of living embryos produced by inter-crossing of <i>p600</i>^+/− mice. The numbers of embryos with indicated genotypes from days E9.5 to E13.5 are shown. Significances of the frequency of survival <i>p600</i>^−/− embryos and expected frequency of 25%, according to Mendelian distribution of the genotypes were calculated by X² test. The <i>p</i>-values of X² test are shown. ** and *** show p-value <0.01 and <0.001, respectively. (C) The appearances of typical embryos of <i>p600</i>^−/− and <i>p600</i>^+/+ littermates from days E9.5 to E12.5. Scale bars indicate 1 mm.</p

Embryonic p600 is required for liver development.

<p>H&E stained transverse sections of <i>p600</i>^−/+ (<i>p600</i>^+/−/<i>Sox2-Cre</i>⁺) (left) and <i>p600</i> cKO (right) liver at days E12.5 (A) and E13.5 (B). Note that <i>p600</i> cKO livers have less densely packed parenchymal hepatocytes. Scale bars indicate 100 µm.</p

Defects in FAK activation in conditional <i>p600</i> knockout heart.

<p>Transverse sections of <i>p600</i>^−/+ (<i>p600</i>^+/−/<i>Sox2-Cre</i>⁺) (left) and <i>p600</i> cKO (right) heart at days E13.5 were stained with anti-desmin (A), anti-FAK (B), anti-phospho-FAK (Tyr397) (C), and anti-MEF2 (D) antibodies. In panels C and D, the position of the inferior atrioventricular endocardial cushions (IC) is labeled. Scale bars indicate 200 µm.</p

Conditional <i>p600</i> knockout specific to embryos.

<p>(A) Conditional deletion strategy of <i>p600</i> allele in embryos. The mice with ‘Neo allele’ were mated with transgenic mice which expresses <i>actin</i> promoter-driven <i>flippase</i>. This crossing eliminated the Neo cassette region between the frt sites (white rectangles), resulting in ‘floxed allele’. After crossing with Tg(<i>Sox2-Cre</i>) mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066269#pone.0066269-Hayashi1" target="_blank">[21]</a>, the region between loxP sites (red triangles) including the first exon of <i>p600</i> is deleted where Cre recombinase is expressed, resulting in ‘KO allele’. (B) Genotype analysis of <i>Sox2-Cre</i>-mediated p600 knockout animals. Male mice of Tg (<i>Sox2-Cre</i>) <i>p600</i>^+/− were crossed with female containing the homozygous <i>p600</i> floxed alleles (<i>p600</i>^flox/flox). Genotypes of resulting littermates were analyzed by PCR. The <i>p</i>-values of X² test are shown. * and ** show significant differences between observed frequency of survival <i>p600</i> cKO embryos and expected frequency of 25%, according to Mendelian distribution of the genotypes with p-score <0.05 and <0.01, respectively. (C) Typical appearances of <i>p600</i> cKO and heterozygous <i>p600</i>^+/− (<i>p600</i>^+/−/<i>Sox2 Cre</i>⁺) embryos at days E12.5 and E13.5. Scale bars indicate 1 mm.</p

Placental abnormalities in <i>p600</i>^−/− animals.

<p>(A) Placental labyrinth defects in <i>p600</i>^−/− animals. H&E staining images of the labyrinth layer of <i>p600</i> knockout and control littermate at E12.5 (the area below the dashed lines) are shown. The labyrinth areas of <i>p600</i>^−/− placenta are thinner than those of wild type littermate as shown by arrows. Scale bars indicate 500 µm. (B) High magnification images of the labyrinth layer. Dilated blood vessels (arrows) are observed in <i>p600</i>^−/− placenta. Scale bars indicate 50 µm. (C) Immunofluorescence staining of blood vessels in labyrinth areas with anti-laminin antibody. The blood vessels in labyrinth are dilated and sparse in <i>p600</i> KO animals at E12.5. Scale bars indicate 100 µm.</p

<i>Sox2-Cre</i>-mediated conditional knockout revealed that embryonic p600 is essential for heart development.

<p>(A) Impaired interventricular septum formation in <i>p600</i> cKO heart. From H&E stained transverse cross-sections of E12.5 embryos, the interventricular septum areas are shown. In the <i>p600</i>^+/− (<i>p600</i>^+/−/<i>Sox2-Cre</i>⁺) heart (left), the interventricular septum (IVS) is fused with inferior atrioventricular endocardial cushions (IC), separating the left and right ventricles. On the other hand, <i>p600</i> cKO heart has developmental defects of the interventricular septum. Scale bars indicate 500 µm. (B) The <i>p600</i>^+/− (left) and <i>p600</i> cKO (right) heart at E13.5. Scale bars indicate 200 µm. (C) Magnified images of ventricular wall. Magnified areas are indicated as boxes in (B). Scale bars indicate 100 µm. (D) Relative cell numbers of the ventricular wall in the <i>p600</i> cKO and <i>p600</i>^+/− hearts at E13.5. Cell numbers of ventricular wall areas were estimated from the several serial sections and shown as relative values. The statistical significance of the difference was calculated with Student’s t-test. * shows <i>p</i>-value <0.05.</p