Primary antibodies.

<p>Primary antibodies.</p

The efficiency of derivation of mESC lines from blastocysts.

<p>The efficiency of derivation of mESC lines from blastocysts.</p

Early embryogenesis occurs normally in p120ctn-depleted embryos.

<p><b>(A-C)</b> p120ctn co-localizes with cadherin-based junctional complexes in wild-type preimplantation mouse embryos. Bright field (BF) transmitted light micrographs and immunofluorescence of wild-type embryos from the two-cell stage to blastocysts, including uncompacted (uncomp.) and compacted (comp.) morulas. Double immunofluorescence was done for p120ctn (green signal) and for E-cadherin <b>(A)</b>, αE-catenin <b>(C)</b> or β-catenin <b>(B)</b>. Scale bars: 25 μm. <b>(D)</b> Breeding scheme to obtain p120ctn^-/- embryos in timed matings. The table depicts numbers and percentages of p120ctn^+/+, p120ctn^+/- or p120ctn^-/- embryos that were recovered at the developmental stages indicated (dpc, days post coitum). <b>(E)</b> BF micrographs and immunostainings of 3.5-dpc blastocysts of wild-type (p120ctn^+/+) mice, heterozygous (p120ctn^+/-) and homozygous (p120ctn^-/-) p120ctn knock-out mice. Double immunofluorescence was performed for p120ctn and E-cadherin. Scale bars: 25 μm. <b>(F)</b> Hematoxylin and eosin (H&E)-stained paraffin sections of control and p120ctn^-/- gastrula-stage embryo. Scale bars: 200 μm.</p

E-cadherin stabilization by p120ctn is required for proper polarization and cell–cell adhesion by EB cells.

<p><b>(A)</b> Confocal pictures of p120ctn and E-cadherin stainings of control and p120ctn^-/-;AL^tg/+ mESCs, and of p120ctn^-/-;AL^tg/+ mESCs expressing various rescue constructs from the R26 promoter, as indicated by the names above each of the images. An 2.9-fold magnified image is shown below each picture. Scale bars: 25 μm. <b>(B)</b> TEM of DIV12 EBs, control or p120ctn^-/-;AL^tg/+ EBs, or p120ctn^-/-;AL^tg/+ EBs expressing various rescue constructs from the R26 promoter, as indicated. Blue boxes depict areas with mature junctions while blue arrows denote areas of minimal cell–cell adhesion. Black scale bars: 10 μm; white scale bars: 1 μm.</p

Primers for genotyping.

<p>Primers for genotyping.</p

qRT-PCR primers.

<p>qRT-PCR primers.</p

Targeted ESC lines generated by RMCE.

<p>Targeted ESC lines generated by RMCE.</p

p120ctn loss affects junctional stability of mESCs, has no effect on self-renewal but partly inhibits differentiation of mESCs.

<p><b>(A)</b> Scheme of isolation of p120ctn^fl/fl mESCs (referred to as control) followed by <i>in vitro</i> Cre-mediated recombination. <b>(B)</b> Scheme of mouse breedings that allow germline Cre-mediated recombination of p120ctn^fl/fl mice <i>in vivo</i>, followed by the isolation of p120ctn^+/+ and p120ctn^+/- mESCs (both referred to as control), and p120ctn^-/- mESCs. <b>(C)</b> Confocal fluorescent images of control and p120ctn-null mESCs stained for p120ctn and E-cadherin. Scale bars: 25 μm. <b>(D)</b> Western blot analysis of two control (p120ctn^fl/fl) and two p120ctn-null mESC lines. Antibodies used were specific for junctional components as indicated. Actin was used as loading control. <b>(E)</b> Morphology, alkaline phosphatase (AP) activity, and Oct4 immunostaining of control and p120ctn-null mESCs. Black scale bars: 100 μm, white scale bars: 50 μm. <b>(F)</b> Control and p120ctn-null EBs were cultured for 30 days <i>in vitro</i> (DIV30, top panels). Thereafter, they were trypsinized, cultured for 4 days in SR-mESC medium, and stained for AP (middle panels). Oct4 immunohistochemistry was performed on paraformaldehyde-fixed paraffin sections of control and p120ctn-null EBs (DIV30, bottom panels). The experiment was performed with two independent control and p120ctn-null mESC lines and was reproduced twice. Scale bars: 200 μm. <b>(G)</b> Fluorescent images showing control and p120ctn-null EB-derived cells, which were plated for 10 days and then stained for a neurectodermal marker (βIII-tubulin), an endodermal marker (α-fetoprotein 1), and three mesodermal markers (CD45, smooth muscle actin/SMA, α-dystrobrevin). This differentiation experiment was performed twice. White scale bars: 200 μm; black scale bar: 200 μm. Pictures <b>(H)</b> and overview table <b>(I)</b> of offspring mice, obtained by diploid embryo aggregation assays with control and p120ctn-null mESCs.</p

Proper expression of p120ctn and E-cadherin is required for endoderm differentiation from mESCs.

<p><b>(A)</b> Immunoprecipitation (IP) experiments for p120ctn-null mESCs with R26-based expression of p120ctn isoform 1A (R1A WT) or its corresponding mutant (R1A K401M). <b>(B)</b> IP experiments for p120ctn-null mESCs with R26-based expression of p120ctn isoform 3A (R3A WT) or its corresponding mutant (R3A K401M). IPs were performed with an anti-p120ctn antibody (IP_Rel) or with an irrelevant anti-GFP antibody (IP_Irrel). Eluates immunoblotted (IB) with an anti-p120ctn antibody (top panels) showed that p120ctn was efficiently bound to the beads. Immunoblotting with an anti-E-cadherin antibody (bottom panels) confirmed the interaction of wild-type p120ctn with E-cadherin, whereas mutated K401M p120ctn was unable to bind E-cadherin. <b>(C)</b> Immunohistochemistry for p120ctn, E-cadherin and AFP on DIV30 p120ctn^-/- EBs expressing from the endogenous R26 promoter either p120ctn isoform 3A (R_p120_3A) or its E-cadherin-uncoupled K401M mutant form (R_p120_3A_K401M). Scale bars: 50 μm. <b>(D)</b> qRT-PCR analysis for endoderm-specific marker genes was performed using cDNAs originating from DIV12 control and p120ctn^-/-;AL^tg/+ EBs, and from p120ctn^-/-;AL^tg/+ EBs expressing various rescue constructs from the R26 promoter as indicated by the EB names. <i>Tbp</i> and <i>Rpl13a</i> were used for normalization. The error bars in the graphs represent the standard deviation of three technical replicates. * or ** denote comparisons that are significantly different. The <i>P</i> values (t test) from left to right are as follows: 0.016, 0.014 and 0.0018.</p