Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: a , A schematic of preimplantation development. ZGA, zygotic genome activation. b , A schematic of differential Hippo signalling in TE (top) and ICM (bottom) lineages in the morula stage mouse embryo. c , A schematic of blastocyst reconstruction assay. Two-cell stage embryos injected with Ezrin –RFP ( Ezrin only, control) or Tfap2c + Tead4 + RhoA mRNA (TTRhoA) were cultured until the early 16-cell stage. Sixteen polarized cells from each genotype were sorted, re-aggregated, cultured until the mid-blastocyst stage and the proportion of ICM examined. d , Representative images of the reconstructed blastocysts from Ezrin -only or TTRhoA embryos. Embryos were immunostained to reveal CDX2 (TE), NANOG (epiblast) and SOX17 (primitive endoderm). e , Quantification of the ratio of ICM from reconstructed blastocysts from Ezrin -only or TTRhoA embryos. The ICM ratio is calculated as the number of cells positive for NANOG or SOX17 divided by the total number of cells (positive for CDX2, NANOG or SOX17). Each dot indicates the data point obtained from one embryo. Data shown as mean ± s.e.m. N = 19 embryos for EZRIN-only group and N = 16 embryos for TTRhoA group. N = 2 experiments. ** P = 0.0266, two-sided Student’s t -test. Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Activation Assay, Injection, Control, Cell Culture

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: ( a ) Representative images of the reconstructed blastocysts from Ezrin-only or TTRhoA embryos. Embryos were immunostained to reveal CDX2 (TE), NANOG (epiblast) and SOX17 (primitive endoderm). Experimental procedures were described as in Fig. . ( b ) Quantifications of TE and ICM cell numbers in each reconstructed blastocyst generated from experiment described in Fig. . * p = 0.0276; ns, not significant, Two-sided student’s t test. Each dot indicates the datapoint obtained from one embryo. N = 19 embryos for Ezrin-only group, N = 16 embryos for TTRhoA group. N = 2 experiments. ( c ) Representative images of embryos injected with Ezrin-RFP or with Tfap2c+Tead4+RhoA mRNA in one cell of the 2-cell stage, and immunostained with DAPI with TFAP2C at the 4–8 cell stage. ( d ) Representative images of embryos injected with Ezrin-RFP or with Tfap2c + Tead4+RhoA mRNA at the one cell of the 2-cell stage, and immunostained with DAPI with TEAD4 at the 8-cell stage. ( e ) Representative images of embryos injected with EZRIN-RFP and dsRNA targeting Tfap2c at the one cell of the 2-cell stage, and immunostained with DAPI with TFAP2C at the 8- stage. ( f ) Representative images of embryos injected with EZRIN-RFP and dsRNA targeting Tead4 at both cells of the 2-cell stage, and immunostained with DAPI with TEAD4 at the 8- stage. N = 2 experiments for c - f . Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Generated, Injection

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: ( a ) Schematic of workflow for experiments in (b-c, e-f). ( b ) Embryos injected with Ezrin-only or also with TTRhoA and analysed at early 8-cell stage for DAPI, CDX2 and EZRIN-RFP. Arrows indicate an injected cell. Quantification shown in c. ( c ) Quantification of normalised CDX2 nuclear signal intensity in Ezrin-only (polar and apolar) cells, TTRhoA polar cells and TTRhoA apolar cells. (Data shown as individual data points with Box and Whisker plots (lower: 25%; upper: 75%; line: median; whiskers: min to max). Each dot indicates an analysed cell. N = 13 embryos for EZRIN-RFP only; n = 15 embryos for TTRhoA group. *p < 0.05, one-way ANOVA test. ( d ) RNA-sequencing analysis of Cdx2 expression level at the 8-cell stage in embryos injected with dsRNA targeting GFP, Tfap2c , Tead4 or Tfap2c and Tead4 (dsTT) at the zygote stage. N = 5 samples for dsGFP and dsTT; n = 4 samples for ds Tfap2c and ds Tead4 . Data is shown as mean ± S.E.M. *p = 0.0159, Kruskal-Wallis test. ( e ) Representative images of CDX2 expression in cells injected with Ezrin-only or also with dsTT at the mid 8-cell stage. Quantification is shown in panel f. ( f ) Quantification of normalised CDX2 nuclear signal intensity. For the normalisation, the nuclear signal of CDX2 was normalised against DAPI, and then the CDX2/DAPI ratio of injected cells were normalised against the value of non-injected cells. Data shown as individual data points with Box and Whisker plots (lower: 25%; upper: 75%; line: median; whiskers: min to max). N = 13 embryos for EZRIN-RFP only; n = 15 embryos for dsTT group. ****p < 0.0001, Mann-Whitney test. Normalisation is as in d. Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Injection, Whisker Assay, RNA Sequencing Assay, Expressing, MANN-WHITNEY

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: a , RNA-sequencing analysis of Gata3 expression level at the eight-cell stage in embryos injected with dsGFP, ds Tfap2c , ds Tead4 or ds Tfap2c + ds Tead4 . N = 5 samples for ds GFP and ds Tfap2c + ds Tead4 and N = 4 samples for ds Tfap2c and ds Tead4 . Data are shown as mean ± s.e.m. * P < 0.05, Kruskal–Wallis test. N = 2 collections. b , A schematic of workflow for experiments in c – h . One blastomere of the two-cell stage embryo was injected with mRNA encoding Ezrin only (as a control), or also with dsRNA targeting Tfap2c and Tead4 , or also with TTRhoA mRNA. c , Representative images of GATA3–GFP expression level in 8–16-cell stage embryos injected with the indicated dsRNA as described in b . Quantifications are shown in d . * P < 0.05, Mann–Whitney test. d , Quantification of the level of GFP in control and embryos injected with dsRNA targeting Tfap2c and Tead4 . * P = 0.0262, Mann–Whitney test. Data are shown as mean ± s.e.m. N = 7 embryos for the Ezrin -only group and N = 7 embryos for the ds Tfap2c + ds Tead4 group. N = 2 experiments. e , Representative images of GATA3–GFP transgenic late eight-cell embryos, after injection with EZRIN only or TTRhoA, as described in b . The arrows indicate an injected cell. The number of embryos and quantifications shown in f . BF, bright field. f , Quantifications of normalized GATA3–GFP signal intensity in the indicated overexpression conditions. For normalization, GFP signal in injected cells were normalized against the noninjected cells. Data are shown as mean ± s.e.m. The numbers indicate the number of embryos analyzed. * P = 0.0218, one-way ANOVA test. g , Representative images of GATA3–GFP expression in embryos injected with EZRIN–RFP mRNA and TTRhoA, as indicated in b , and treated with water (control) or C3-transferase (RhoA inhibitor) at the late eight-cell stage. Quantifications are shown in h . h , A time course of t h e normalized GATA3–GFP signal intensity in cells overexpressing EZRIN–RFP only (control), or also exposed to TTRhoA, RhoA inhibitor or TTRhoA + RhoA inhibitor. Data are shown as mean ± s.e.m. n = 7 embryos for each group. The yellow region indicates the early stages of developmental when Gata3 expression is insensitive to RhoA activity (before the 16-cell stage) and the purple region indicates RhoA-sensitive stages (after the 16-cell stage). Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: RNA Sequencing Assay, Expressing, Injection, Control, MANN-WHITNEY, Transgenic Assay, Over Expression, Activity Assay

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: ( a ) Snapshots of GATA3-GFP transgenic embryos injected with EZRIN-RFP at different developmental stages. GFP signal was undetectable until 16- cell stage, when it was expressed in all cells. Segregation of GFP to the outer polarised cells is observed from 16–32 cell stage. n = 35 embryos examined. Scale bars, 15 μm. ( b ) Representative images of 4-, late 8-, 16- and 32–64 cell stage embryos carrying GATA3-GFP transgene to immunostain with DAPI and GATA3. A similar expression profile in time and space is detected between the signals yielded from GATA3-GFP transgene reporter and GATA3 antibody. N = 2 embryos for each stage. Scale bars, 15 μm. ( c ) Single cell genotyping of cells that show high (Cas9 only group) and low (Cas9+sgRNAs) levels of TFAP2C and TEAD4 protein. Criteria for cell grouping is as previously described.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Transgenic Assay, Injection, Expressing

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: a , The expression of Nanog , Pou5f1 ( Oct4 ) and Klf5 by bulk RNA sequencing in the indicated conditions. ** P = 0.0042 for Nanog , P = 0.0021 for Pou5f1 and P = 0.0019 for Klf5 , Kruskal–Wallis test. N = 5 samples for ds GFP and ds Tfap2c + ds Tead4 and N = 4 samples for ds Tfap2c and ds Tead4 . N = 2 collections. Data are shown as mean ± s.e.m. b , Representative images of embryos injected with Cas9 mRNA or with gRNAs targeting Tfap2c gene locus, to fix at the mid eight-cell stage and stain for NANOG and TFAP2C. The quantification is shown in c . c , Quantification of NANOG expression in Cas9-only (control) or Tfap2c -depleted cells by CRISPR–Cas9 shown in b . **** P < 0.0001, two-sided Student’s t -test. N = 27 embryos for the Cas9-only group and N = 10 embryos for Tfap2c KO embryos. N = 2 experiments. Data are shown as individual data points with box and whisker plots (bottom: 25%; upper: 75%; line: median; whiskers: min to max). d , Representative images of embryos injected with EZRIN–RFP mRNA alone or with Tfap2c and Tead4 mRNA, and visualized NANOG expression at the mid eight-cell stage. The embryos were injected at the two-cell stage and fixed at the mid eight-cell stage. e , Quantification of NANOG protein levels in conditions showing in d . N = 39 cells for the EZRIN–RFP group and N = 17 cells for the Tfap2c + Tead4 group. N = 2 experiments. **** P < 0.0001, Mann–Whitney test. Data are shown as individual data points with box and whisker plot (bottom: 25%; upper: 75%; line: median; whiskers: min to max). f , Representative images of embryos injected with EZRIN–RFP mRNA alone or with Tfap2c and Tead4 mRNA at one cell of the two-cell stage and fixed at the early to mid eight-cell stage, and visualized POU5F1 (OCT4) expression at the mid eight-cell stage. g , Quantification of OCT4 protein levels in cells from conditions shown in f . N = 28 cells for the Ezrin –RFP group and N = 15 cells for the Tfap2c + Tead4 group. N = 2 experiments and **** P < 0.0001, Mann–Whitney test. Data are shown as individual data points with box and whisker plots (bottom: 25%; upper: 75%; line: median; whiskers: min to max). Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Expressing, RNA Sequencing Assay, Injection, Staining, Control, CRISPR, Whisker Assay, MANN-WHITNEY

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: a , The expression profile of Amot , Amotl2 and Lats2 , data obtained from Deng et al. b , The expression of Amot (*** P = 0.0052, Kruskal–Wallis test), Amotl2 (* P = 0.0115, one-way ANOVA) and Lats2 (**** P < 0.0001, Kruskal–Wallis test) by bulk RNA sequencing in the indicated conditions. The expression level is shown as mean ± s.e.m. N = 5 samples for ds GFP and ds Tfap2c + ds Tead4 and N = 4 samples for ds Tfap2c and ds Tead4 . N = 2 collections. c , Late eight-cell embryos injected with EZRIN–RFP + dsGFP (control) or EZRIN–RFP + ds Tfap2c in half embryo and immunostained AMOT, EZRIN–RFP and DNA (DAPI). BF, bright field. d , Quantification of plasma membrane-localized AMOT as in c . N = 17 cells for EZRIN–RFP and N = 14 cells for ds Tfap2c . N = 2 experiments. ** P = 0.0087, two-sided Mann–Whitney test. e , Mid-eight-cell embryos injected with Cas9 mRNA or with Tfap2c sgRNAs stained with TFAP2C and AMOT. f , Quantification of membrane AMOT as in e . N = 62 cells for Cas9 only and N = 18 cells for Tfpa2c CRISPR. N = 2 experiments. **** P < 0.0001, two-sided Mann–Whitney test. g , Late four-cell embryos overexpressing EZRIN–RFP or with Tfap2c in half embryo immunostained with AMOT, EZRIN–RFP and DNA (DAPI). h , Quantification of membrane AMOT as in k . N = 11 embryos for EZRIN and N = 9 embryos for the Tfap2c group. N = 2 experiments. ** P = 0.0013, two-sided Mann–Whitney test. i, Late eight-cell embryos i njected with dsRNA targeting GFP (control) or Tead4 dsRNA in half embryo and immunostained EZRIN–RFP and p-YAP. j , Quantification of cytoplasmic p-YAP levels as in g . *** P < 0.001, Mann–Whitney test. N = 8 embryos for dsGFP and N = 8 embryos for ds Tead4 . N = 2 experiments. k , Mid eight-cell embryos injected with Cas9 mRNA or with Tead4 sgRNAs stained with TEAD4 and p-YAP. l , Quantification of cytoplasmic p-YAP levels as in i . N = 87 cells for Cas9 and N = 68 cells for Tead4 CRISPR. N = 2 experiments. **** P < 0.0001, two-sided Mann–Whitney test. m , Mid eight-cell embryos overexpressing EZRIN–RFP or with Tead4 in half embryo immunostained EZRIN–RFP and p-YAP. n , Quantification of cytoplasmic p-YAP as in m . N = 10 embryos for EZRIN and N = 16 embryos for the Tead4 group. N = 2 experiments. *** P = 0.0002, two-sided Mann–Whitney t -test. For d , f , h , j , l and n , data are shown as individual data points with box and whisker plots (bottom: 25%; upper: 75%; line: median; whiskers: min to max). Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Expressing, RNA Sequencing Assay, Injection, Control, Membrane, MANN-WHITNEY, Staining, CRISPR, Whisker Assay

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: ( a ) Representative images of embryos microinjected with EZRIN-RFP mRNA alone, or with Tfap2c + Tead4 mRNA, or Klf5 siRNA, or Tfa2pc + Tead4 mRNA+ Klf5 siRNA in one cell of the 2-cell stage, and to immunostain for KLF5, CDX2 and DAPI. ( b ) Quantification of KLF5 protein levels in conditions showing in a. KLF5 siRNA effectively eliminated KLF5 expression at the protein level, Tfap2c and Tead4 overexpression had no effect on KLF5’s expression. ( c ) Quantification of CDX2 protein levels in conditions shown in a. Tfap2c and Tead4 expression induced significant upregulation of CDX2 expression at the protein level regardless of the depletion of KLF5. ****p < 0.0001, ns, not significant, Kruskal-Wallis’ test. For b and c, data are shown as bar chart with mean ± SEM and individual data points. N = 19 embryos from Ezrin only group; N = 12 embryos from Tfap2c+Tead4 group; =10 embryos from siKlf5 group; N = 11 embryos from Tfap2c+Tead4+siKlf5 group. N = 2 experiments. ( d ) The expression profile of Nf2 , Mob1 , Yap1 , Wwtr1 , Amotl1 and Lats1 throughout preimplantation development. Data obtained from Deng et al. . ( e ) Representative images of embryos microinjected with EZRIN-RFP mRNA alone, or with Tfap2c + Tead4 mRNA, or Tfa2pc + Tead4 mRNA+ Klf5 siRNA in one cell of the two-cell stage, and immunostained for AMOT and DAPI. Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Expressing, Over Expression

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: a , The mRNA expression profiles of TFAP2C and TEAD4 in preimplantation human embryos. Data retrieved from Stirparo et al., 2018 (ref. ). Data are shown as mean ± s.e.m. b , Human embryos before and after polarization were fixed and stained for PARD6 and TFAP2C. N = 4 embryos were examined. c – f , mRNA expression profile of Gata3/GATA3 ( c ), Amot/AMOT ( d ), Amotl2/AMOTL2 ( e ) and Lats2/LATS2 ( f ) in preimplantation mouse and human embryos. Data retrieved from Stirparo et al., 2018 (ref. ). g , The University of California, Santa Cruz browser view showing accessible chromatin regions in Gata3/GATA3 and Amot/AMOT , Amot l2 /AMOTL2 and Lats2/LATS2 loci in the mouse and human embryos at different stages, determined from ATAC-sequencing data. Mouse data retrieved from Wu et al. . Human data retrieved from Wu et al. . Scale bars, 15 μm.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Expressing, Staining, Sequencing

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: ( a ) DNA sequence of the enhancer, promoter and the intron regions of mouse and human Gata3/GATA3 locus was analysed and accessible TFAP2C/TEAD4 binding sites were predicted. Sites with DNA sequence homology above 50% were considered as conserved. ( b ) DNA sequence of the enhancer, promoter and the intron regions of mouse and human and Amot/AMOT , Amotl2/AMOTL2 , Lats2/LAST2 were analysed and accessible TFAP2C/TEAD4 binding sites were predicted. DNA sequence homology above 50% were considered as conserved.

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Sequencing, Binding Assay

Journal: Nature Structural & Molecular Biology

Article Title: Tead4 and Tfap2c generate bipotency and a bistable switch in totipotent embryos to promote robust lineage diversification

doi: 10.1038/s41594-024-01311-9

Figure Lengend Snippet: In the bipotential state YAP is both nuclear (as in the TE) and cytoplasmic (as in the ICM). We propose here that zygotically expressed Tead4 and Tfap2c accumulate in the nucleus and promote multilineage priming (expression of ICM- and TE-specific transcription factors and of Hippo components). Hippo signaling is intermediate (gray) (active AMOT, AMOTL2 and LATS2 in the cytoplasm), coincident with a preliminary apical domain. At the 16-cell stage, differential Hippo signaling mediated by Tead4 , Tfap2c and RhoA and the TE-specific apical domain leads to diversification of the lineages: TE (blue) with Hippo OFF and ICM (beige) with Hippo ON. In the absence of Tfap2c and Tead4 , no apical domain is formed and the transient TE/ICM composite state is abolished. As a result, the cells obtain an ICM-like state due to the low but phosphorylated cytoplasmic YAP, as well as the low-level expression of ICM fate specifiers. In the absence of the Hippo system, the cell fate specification is retarded and the cells are trapped in a null state without TE or ICM specification. We propose that totipotency diminishes to bipotency after zygotic genome activation initiates expression of Tead4 and Tfap2c .

Article Snippet: The primary antibodies were as follows: rabbit polyclonal anti-Pard6b (Santa Cruz, sc-67393, 1:200), mouse monoclonal anti-GFP (Nacalai Tesque Inc., 04404-84, 1:500), mouse monoclonal anti-Tfap2c (Santa Cruz, sc-12762, 1:200), goat monoclonal anti-Tfap2c (R&D Systems, AF5059-SP, 1:200), rabbit monoclonal anti-Tead4 (Abcam, ab97460, 1:200), mouse monoclonal anti-Tead4 (Abcam, ab58310, 1:100), goat monoclonal anti-Sox17 (R&D Systems, af1924), mouse monoclonal anti Cdx2 (Biogenex, MU392-UC, 1:200), rabbit monoclonal anti-Nanog (Abcam, ab80892, 1:200), rabbit monoclonal anti-phosphorylated-Yap (Cell Signaling Technologies, 4911S, 1:200), mouse monoclonal anti-Yap (Santa Cruz, sc-101199, 1:200), goat polyclonal anti-Amot (Santa Cruz, sc-82491, 1:1,000), and rabbit polyclonal anti-Klf5 (Proteintech, 21017–1-AP).

Techniques: Expressing, Activation Assay