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SC_11-0927_sm_SupplFig1ABC.tiff528KSupplemental Figure 1. Targeting of the ZO-1 locus and genotyping. A. Targeting strategy. Schematic representation of the genomic locus of ZO-1 showing exon 1 with the initiation ATG (red box). The targeting vector is designed to disrupt exon 1 by the in-frame insertion of a lacZ gene (green box) and a loxP-flanked neomycin cassette (blue circles and white box, respectively) immediately downstream of the ATG codon. Red bars indicate the regions to which probes used for Southern blot analysis hybridize; black arrows denote the regions where primers used for genotyping anneal. B. Homologous recombination in mESCs. Southern blot of ScaI-digested genomic DNA of selected mESC clones hybridized with labeled DNA probe hybridizing to a 5′ genomic DNA probe for the identification of homologous recombinants. ScaI fragments of 10 kb and 14.8 kb corresponding to the WT and targeted mutant alleles, respectively, are detected with the 5- genomic probe in heterozygous mESC clones (+/-), whereas only the fragment corresponding to the WT or the mutant allele is present in controls (+/+) or ZO-1 KO (−/−) clones, respectively. C. Western blot detection of ZO-1. Lysates of two independent ZO-1+/+, ZO-1+/- and ZO1−/− mESC clones each were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to membranes, and blotted with antibodies to ZO-1.
SC_11-0927_sm_SupplFig2AB.pdf278KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2C.pdf296KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2D.tiff1848KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2EFG.tiff1350KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2H.pdf292KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2IJ.pdf269KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2KL.tiff2936KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2MN.pdf245KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig2O.tiff105KSupplemental Figure 2. Characterization of mESCs. A-C. mESC colony morphology and AP expression. ZO-1+/+ or ZO-1−/− mESCs were cultured in defined N2B27 media supplemented with LIF and Bmp4 for 6 days (panel A) or in media supplemented with LIF and FBS for 7 days (panel C). Bar: 200 μm. Phase contrast images were acquired after AP staining in (A) and (C). Quantitation of the number of mESC clones that show outgrowth of differentiated cells is shown in (B). D. Analysis of pluripotency and differentiation marker expression by immunofluorescence microscopy. ZO-1+/+ (panels a-c) and ZO-1−/− (panels d-f) mESC colonies were fixed, permeabilized, and stained with antibodies to Nanog (a and d; red color) and GATA4 (b and e; green color). Panel c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. E, F. Quantification of the expression levels of Gata4. Bands from Western blot analysis (E) were scanned, the density of (+/+) at D2 set to one and relative expression levels for the other conditions calculated and plotted (F). GAPDH served as loading control. The data shown is the mean ± SEM of three independent experiments as shown in (B). * P<0.05, Student's t-test. G. GATA4 positive cell are not apoptotic. ZO-1+/+ mESC colonies were fixed, permeabilized, and stained for Tunnel reactivity (a; red color) and antibodies for GATA4 (b; green color). Panel c shows the merged image with the nuclei labeled with DAPI (blue color). Bar: 50 μm. H. Outgrowth of differentiated cells in Go Germline and TT2 mESCs. The indicated mESC lines were cultured for 6 days in media supplemented with LIF and Bmp4 and phase contrast images were acquired. Bar: 200 μm. I. Pluripotency and differentiation marker expression in different mESC lines. Expression of the indicated genes in the different mESC lines was assessed by semi-quantitative RT-PCR. Actin served as a loading control. J. Long term culture ZO-1+/+ and ZO-1−/− mESCs. Phase contrast images of mESC cultures after 9 passages in media with LIF and FBS. K. Chimera derived from W4 mESCs. W4 mESCs cultured in N2B27 media supplemented with LIF and Bmp4 generated chimera with a high percentage Agouti coat color (2 mice on the left). L. Outgrowth of differentiated cells is absent in ZO-1−/− mESCs despite the lack of apoptosis. ZO-1+/+ or ZO-1−/− mESCs were cultured for 4 days in media supplemented with LIF only, phase contrast images were acquired (Fig. 2D) and number of mESC colonies with outgrowth of differentiated cells quantitated. M. Cell density dependent apoptosis in ZO-1−/− mESC cultures. 10x104 or 150x104 mESCs lacking ZO-1 were cultured for 4 days in N2B27 media supplemented with LIF and Bmp4. Apoptosis was monitored by detecting activated Caspase 3 levels by Western blot analysis. Dab2 levels were detected to assess differentiation. GAPDH served as loading control. N. Phase contrast images of ZO-1−/− mESCs cultured for 2 days in N2B27 supplemented with LIF and Bmp4. Bar: 200 μm. O. Colony formation. Control or ZO-1−/− mESCs were cultured in N2B27 media with LIF and Bmp4 for 6 days, trypsinized and the 500 cells each plated in media with LIF and FBS and the number of colonies formed were counted and plotted.
SC_11-0927_sm_SupplFig3AB.tiff229KSupplemental Figure 3. The Mek/Erk pathway is repressed in ZO-1−/− mESCs. A. The Mek inhibitor PD98059 phenocopies the lack of ZO-1. The number of mESC colonies with outgrowth of differentiated cells was quantitated from phase contrast images of ZO-1+/+ or ZO-1−/− mESC colonies cultured for 6 days in N2B27 media supplemented with LIF and Bmp4, either in the absence or presence of PD58059 (Fig. 3A). B. Exogenous FGF4 rescues the lack of spontaneous differentiation of ZO-1−/− mESCs. The number of mESC colonies with outgrowth of differentiated cells was quantitated from ZO-1−/− mESC colonies cultured for 6 days in N2B27 media supplemented with LIF and Bmp4, either in the absence or presence of exogenous FGF4 (Fig. 3D). C. FGF4 induced differentiation of ZO-1−/− mESCs is abrogated by the Mek inhibitor PD98059 or the FGFR inhibitor Su5402. Phase contrast images of ZO-1−/− mESC colonies cultured for 6 days in N2B27 media supplemented with LIF and Bmp4 either in the absence of additives (panel a), or in the presence of FGF4 (panel b), FGF4 + PD98058 (panel c) or FGF4 + Su5402 (panel d). Bar: 200 μm.
SC_11-0927_sm_SupplFig3C.pdf301KSupplemental Figure 3. The Mek/Erk pathway is repressed in ZO-1−/− mESCs. A. The Mek inhibitor PD98059 phenocopies the lack of ZO-1. The number of mESC colonies with outgrowth of differentiated cells was quantitated from phase contrast images of ZO-1+/+ or ZO-1−/− mESC colonies cultured for 6 days in N2B27 media supplemented with LIF and Bmp4, either in the absence or presence of PD58059 (Fig. 3A). B. Exogenous FGF4 rescues the lack of spontaneous differentiation of ZO-1−/− mESCs. The number of mESC colonies with outgrowth of differentiated cells was quantitated from ZO-1−/− mESC colonies cultured for 6 days in N2B27 media supplemented with LIF and Bmp4, either in the absence or presence of exogenous FGF4 (Fig. 3D). C. FGF4 induced differentiation of ZO-1−/− mESCs is abrogated by the Mek inhibitor PD98059 or the FGFR inhibitor Su5402. Phase contrast images of ZO-1−/− mESC colonies cultured for 6 days in N2B27 media supplemented with LIF and Bmp4 either in the absence of additives (panel a), or in the presence of FGF4 (panel b), FGF4 + PD98058 (panel c) or FGF4 + Su5402 (panel d). Bar: 200 μm.
SC_11-0927_sm_SupplFig5A.tiff131KSupplemental Figure 5. Differentiation of ZO-1−/− mESCs is delayed upon LIF/Bmp4 withdrawal. A. The number of mESC colonies with outgrowth of differentiated cells was quantitated from phase contrast images of WT control or ZO-1−/− mESC colonies cultured for 2 (D2) or 6 (D6) days in the absence of LIF and Bmp4 (Fig. 5A). B. Expression of pluripotency and differentiation markers following LIF and Bmp4 withdrawal detected by immunofluorescence microscopy. ZO-1+/+ (panels a-c) or ZO-1−/− (panels d-f) mESCs cultured in the absence of LIF and Bmp4 for 6 days were fixed, permeabilized and stained with antibodies to Oct4 (a and d; green color) and Troma-1 (b and e; red color). Panels c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 20 μm.
SC_11-0927_sm_SupplFig5B.tiff2513KSupplemental Figure 5. Differentiation of ZO-1−/− mESCs is delayed upon LIF/Bmp4 withdrawal. A. The number of mESC colonies with outgrowth of differentiated cells was quantitated from phase contrast images of WT control or ZO-1−/− mESC colonies cultured for 2 (D2) or 6 (D6) days in the absence of LIF and Bmp4 (Fig. 5A). B. Expression of pluripotency and differentiation markers following LIF and Bmp4 withdrawal detected by immunofluorescence microscopy. ZO-1+/+ (panels a-c) or ZO-1−/− (panels d-f) mESCs cultured in the absence of LIF and Bmp4 for 6 days were fixed, permeabilized and stained with antibodies to Oct4 (a and d; green color) and Troma-1 (b and e; red color). Panels c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 20 μm.
SC_11-0927_sm_SupplFig6AB.tiff240KSupplemental Figure 6. Differentiation of ZO-1−/− mESCs is delayed upon LIF/Bmp4 withdrawal. A. LIF and Bmp4 withdrawal does not alter ZO-1 experssion. ZO-1+/+ mESCs cultured in the absence of LIF and Bmp4 were harvested after 0-3 days in culture, lysed and subjected to Western blot analysis using antibodies to ZO-1. GAPDH served as a loading control. B. mESC colony morphology. The number of mESC colonies with outgrowth of differentiated cells was quantitated from phase contrast images of WT control or ZO-1−/− mESC colonies cultured for 3 days in the presence of Bmp4 (Fig. 6A). C. Neural differentiation. Control and ZO-1−/− mESCs were cultured for 10 days in N2B27 media without LIF and Bmp4 to drive differentiation into the neural lineage. Cells were then fixed, permeabilized and stained with antibodies to the neural marker β3-tubulin (a and d; green color) and Troma-1 (b and e; red color). Panels c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. D. Cardiac differentiation. Control and ZO-1−/− mESCs were as detailed in Materials and Methods to facilitate differentiation into the cardiac lineage. Cells were then fixed, permeabilized and stained with antibodies to the cardiac marker MF20 (a and b; red color) and DAPI (d and e; blue color). Bar: 100 μm.
SC_11-0927_sm_SupplFig6C.pdf312KSupplemental Figure 6. Differentiation of ZO-1−/− mESCs is delayed upon LIF/Bmp4 withdrawal. A. LIF and Bmp4 withdrawal does not alter ZO-1 experssion. ZO-1+/+ mESCs cultured in the absence of LIF and Bmp4 were harvested after 0-3 days in culture, lysed and subjected to Western blot analysis using antibodies to ZO-1. GAPDH served as a loading control. B. mESC colony morphology. The number of mESC colonies with outgrowth of differentiated cells was quantitated from phase contrast images of WT control or ZO-1−/− mESC colonies cultured for 3 days in the presence of Bmp4 (Fig. 6A). C. Neural differentiation. Control and ZO-1−/− mESCs were cultured for 10 days in N2B27 media without LIF and Bmp4 to drive differentiation into the neural lineage. Cells were then fixed, permeabilized and stained with antibodies to the neural marker β3-tubulin (a and d; green color) and Troma-1 (b and e; red color). Panels c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. D. Cardiac differentiation. Control and ZO-1−/− mESCs were as detailed in Materials and Methods to facilitate differentiation into the cardiac lineage. Cells were then fixed, permeabilized and stained with antibodies to the cardiac marker MF20 (a and b; red color) and DAPI (d and e; blue color). Bar: 100 μm.
SC_11-0927_sm_SupplFig6D.pdf192KSupplemental Figure 6. Differentiation of ZO-1−/− mESCs is delayed upon LIF/Bmp4 withdrawal. A. LIF and Bmp4 withdrawal does not alter ZO-1 experssion. ZO-1+/+ mESCs cultured in the absence of LIF and Bmp4 were harvested after 0-3 days in culture, lysed and subjected to Western blot analysis using antibodies to ZO-1. GAPDH served as a loading control. B. mESC colony morphology. The number of mESC colonies with outgrowth of differentiated cells was quantitated from phase contrast images of WT control or ZO-1−/− mESC colonies cultured for 3 days in the presence of Bmp4 (Fig. 6A). C. Neural differentiation. Control and ZO-1−/− mESCs were cultured for 10 days in N2B27 media without LIF and Bmp4 to drive differentiation into the neural lineage. Cells were then fixed, permeabilized and stained with antibodies to the neural marker β3-tubulin (a and d; green color) and Troma-1 (b and e; red color). Panels c and f show the merged images with the nuclei labeled with DAPI (blue color). Bar: 100 μm. D. Cardiac differentiation. Control and ZO-1−/− mESCs were as detailed in Materials and Methods to facilitate differentiation into the cardiac lineage. Cells were then fixed, permeabilized and stained with antibodies to the cardiac marker MF20 (a and b; red color) and DAPI (d and e; blue color). Bar: 100 μm.

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