Fig 1.
E-cadherin-null mESCs fail to maintain cellular aggregation and show defects in differentiation.
(A) Scheme of the isolation and Cre-mediated recombination of Ecadhfl/fl mESCs leading to E-cadherin-null mESCs. (B) Morphology and immunostaining for E-cadherin and p120ctn of E-cadherin-floxed (Ecadhfl/fl, control) and E-cadherin-null (Ecadh-/-) mESCs that were cultured on mitomycin-C-treated mouse embryonic fibroblasts (MEFs) and in serum-containing medium. E-cadherin loss resulted in defective mESC aggregation and in cytoplasmic p120ctn levels. An eight-fold magnification of the boxed area is shown in the bottom panels. Scale bars: 50 μm. (C) Morphology and alkaline phosphatase (AP) staining of control and E-cadherin-null mESCs that were cultured in SR-mESC medium supplemented with 2i. White scale bars: 100 μm. Black scale bars: 25 μm. (D) Embryoid bodies (EBs) from control and E-cadherin-null mESCs were cultured for 30 days in vitro (DIV30, top panel). Thereafter, EBs were trypsinized, and an equal number of dissociated cells was cultured for 4 days in SR-mESC medium and then stained for AP (bottom panel). Inset: morphology of dissociated EB cells after replating and AP staining. E-cadherin-null mESCs failed to form EBs and retained AP activity. White scale bars: 200 μm. Black scale bars: 100 μm. (E) Fluorescent images showing control and E-cadherin-null EB-derived cells that were plated for 10 days and then stained for Oct4 (stem cell marker), nestin (neurectoderm), skeletal muscle marker α-dystrobrevin (mesoderm), smooth muscle actin (SMA, mesoderm) and α-fetoprotein 1 (endoderm). Cultures derived from E-cadherin-null mESCs failed to exit their self-renewal and did not commit towards differentiation. Scale bars: 100 μm.
Fig 2.
Early embryogenesis occurs normally in p120ctn-depleted embryos.
(A-C) 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 (A), αE-catenin (C) or β-catenin (B). Scale bars: 25 μm. (D) 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). (E) 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. (F) Hematoxylin and eosin (H&E)-stained paraffin sections of control and p120ctn-/- gastrula-stage embryo. Scale bars: 200 μm.
Fig 3.
p120ctn loss affects junctional stability of mESCs, has no effect on self-renewal but partly inhibits differentiation of mESCs.
(A) Scheme of isolation of p120ctnfl/fl mESCs (referred to as control) followed by in vitro Cre-mediated recombination. (B) Scheme of mouse breedings that allow germline Cre-mediated recombination of p120ctnfl/fl mice in vivo, followed by the isolation of p120ctn+/+ and p120ctn+/- mESCs (both referred to as control), and p120ctn-/- mESCs. (C) Confocal fluorescent images of control and p120ctn-null mESCs stained for p120ctn and E-cadherin. Scale bars: 25 μm. (D) Western blot analysis of two control (p120ctnfl/fl) and two p120ctn-null mESC lines. Antibodies used were specific for junctional components as indicated. Actin was used as loading control. (E) Morphology, alkaline phosphatase (AP) activity, and Oct4 immunostaining of control and p120ctn-null mESCs. Black scale bars: 100 μm, white scale bars: 50 μm. (F) Control and p120ctn-null EBs were cultured for 30 days in vitro (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. (G) 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 (H) and overview table (I) of offspring mice, obtained by diploid embryo aggregation assays with control and p120ctn-null mESCs.
Fig 4.
A structure-function based screening scrutinizes the p120ctn domains required for cystic EB formation.
(A) Morphology (H&E-staining, top panels), ultrastructure (TEM, middle panels) and immunohistochemistry for E-cadherin (bottom panels) of control and p120ctn-null DIV30 EBs. Boxed area in H&E pictures is magnified to visualize the single-layered endodermal layer (arrow). The dashed line in the TEM micrographs separates the endodermal cells (arrows) from the inner EB cells (asterisk). Scale bars: 10 μm. (B) Breeding strategy and mESC isolation procedure to obtain p120ctn-/- mESCs that harbor a recombination-mediated cassette exchange (RMCE)-compatible anti-Luc (AL) allele in the ROSA26 (R26) locus. (C) Micrographs showing DIV30 control and p120ctn-null EBs. Scale bars: 200 μm. (D) Schematic representation of coding potential of key rescue constructs that were introduced into the R26 locus of p120ctn-/-;ALtg/+ mESCs. See Table 1 for the complete list of rescue constructs. p120ctn isoforms contain a central armadillo domain consisting of nine armadillo repeats (grey boxes), one out of four alternative start codons (arrows), and possibly sequences encoded by alternatively used exons A and C (black boxes), and including or excluding two Rho GTPase binding domains (RBDs). p120ctn isoforms 1A, 3A and 4A use the first, third and fourth translation initiation site, respectively. The position of mutations of important AA and the artificial addition of a membrane-targeting motif (CAAX) are shown. E-cadherin (Ecadh) consists of a signal peptide (S), a pro-domain (PRO), four extracellular cadherin (EC) repeats, a membrane-proximal extracellular domain (MPED), a transmembrane domain (T), a juxtamembrane domain (JMD) and a β-catenin-binding domain (CBD). (E-H) Micrographs of DIV30 EBs after introduction of various rescue constructs by RMCE of mESCs, as indicated by the names on top of the images. Scale bars: 200 μm.
Table 1.
Targeted ESC lines generated by RMCE.
Fig 5.
E-cadherin stabilization by p120ctn is required for proper polarization and cell–cell adhesion by EB cells.
(A) Confocal pictures of p120ctn and E-cadherin stainings of control and p120ctn-/-;ALtg/+ mESCs, and of p120ctn-/-;ALtg/+ 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) TEM of DIV12 EBs, control or p120ctn-/-;ALtg/+ EBs, or p120ctn-/-;ALtg/+ 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.
Fig 6.
Proper expression of p120ctn and E-cadherin is required for endoderm differentiation from mESCs.
(A) Immunoprecipitation (IP) experiments for p120ctn-null mESCs with R26-based expression of p120ctn isoform 1A (R1A WT) or its corresponding mutant (R1A K401M). (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. (C) 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. (D) qRT-PCR analysis for endoderm-specific marker genes was performed using cDNAs originating from DIV12 control and p120ctn-/-;ALtg/+ EBs, and from p120ctn-/-;ALtg/+ EBs expressing various rescue constructs from the R26 promoter as indicated by the EB names. Tbp and Rpl13a 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 P values (t test) from left to right are as follows: 0.016, 0.014 and 0.0018.
Table 2.
Primers for genotyping.
Table 3.
The efficiency of derivation of mESC lines from blastocysts.
Table 4.
Primary antibodies.
Table 5.
qRT-PCR primers.