Weidgang CE , Russell R , Tata PR , Kühl SJ , Illing A , Müller M , Lin Q , Brunner C , Boeckers TM , Bauer K , Kartikasari AE , Guo Y , Radenz M , Bernemann C , Weiß M , Seufferlein T , Zenke M , Iacovino M , Kyba M , Schöler HR , Kühl M , Liebau S , Kleger A .

	Figure 1. TBX3 Expression in Developing Mouse and Xenopus Embryos(A) Tbx3 (blue) in preimplantation embryos plotted with other mesendoderm class ESC genes (Oct4, red; Klf5, brown; Klf9, pink; T, violet). Data were obtained upon reanalysis of published data sets.(B) IF of E3.5 blastocyst for OCT4 (red) and TBX3 (green). Scale bars, 20 μm. Nuclei in DAPI (blue).(C–E) TBX3 expression in postimplantation mouse embryos. (C) Tbx3 mRNA in situ hybridization at E6.25 (prestreak) and E7.5 (midstreak). exVE, extraembryonic visceral endoderm. Arrows depict Tbx3-positive cells within the proximoposterior epiblast at E6.25 and mesoderm at E7.5. (D) Tbx3 mRNA in whole mounts of wild-type embryos at E7.75 and E8.25. (E) Z-section of an E6.5 TBX3-GFP reporter embryo. Nuclei in DAPI (blue). Scale bars, 100 μm.(F and G) tbx3 expression in Xenopus. (F) Temporal expression of tbx3 during early Xenopus embryogenesis. tbx3 transcripts are maternally supplied. Zygotic expression starts at stage 10 and increases during gastrulation. gapdh as loading control. Negative control represents a −RT reaction with gapdh. (G) Spatial expression of tbx3 at stages indicated during Xenopus development. tbx3 is detected in the invaginating mesoderm during gastrulation. White arrowheads, tbx3-positive ectodermal cells; red arrowheads, tbx3 expression in the developing cardiac region. m, mesoderm; cm, cardiac mesoderm. Scale bars, 0.5 mm.See also Figure S5.
	Figure 2. TBX3 Expression in Differentiating ESCs(A–C) TBX3 expression during EB-based ESC differentiation. (A) qPCR analysis; (B) immunoblot and quantification of three independent experiments; (C) whole-mount IF of TBX3 in differentiating EBs.(D–G) Expression of lineage-specific markers (EOMES, T, SOX17, and MESP1) during ESC differentiation: (D) gene expression; (E–G) protein expression.(H) IF shows coexpression of TBX3 with EOMES (upper panel row), T (middle panel row), and SOX17 (lower panel row) in differentiating ESCs (day 4). Quantifications of counted cells as bar graphs on the right.(I) FACS of T-GFP/DPP4-RFP knockin ESCs on day 4 of differentiation; inset, description of reporter alleles.(J) Tbx3 gene expression in cell populations outlined in (I).(K) FACS plot of SOX17-RFP knockin ESCs on day 4 of differentiation; inset shows description of reporter alleles.(L) Tbx3 expression in cell populations outlined in (K); pluripotent cells were depleted by SSEA1 staining.(M) TBX3 immunostaining (magenta) of FACS sorted T-GFP-positive and DPP4-RFP-negative cells.(N) TBX3 immunostaining (magenta) of SOX17-RFP-positive and SSEA1-negative cells. Scale bars, 20 μm. Nuclei in DAPI (blue). Error bars = SEM. n = 3 for all experiments in biological replicates.∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S1.
	Figure 3. TBX3 Promotes Mesoderm and Endoderm(A) Illustration of the differentiation protocol. EBs were generated over time from pluripotent ESCs at day 0 (orange) and markers from all three germ layers (ectoderm, blue; mesoderm, yellow; endoderm, green) were generated in –Dox samples, whereas mesoderm and endoderm were upregulated in the +Dox condition.(B) Whole-mount IF in iTBX3 EBs (day 4) for Eomes (left panel), T (middle panel), and Sox17 (right panel) (all in red) upon TBX3 induction (±Dox). All scale bars are 20 μm. Nuclei in DAPI (blue).(C–E) qPCR analysis showing expression of marker genes at day 4 of differentiation: (C) mesendoderm, (D) mesoderm and endoderm, (E) trophoblast and ectoderm upon TBX3 induction via doxycycline (±Dox). n = 3 for all experiments in biological replicates.(F) TBX3 shRNA shows reduced levels of Tbx3, T, and Eomes expression levels already at EB day 1 after a 4 day feeder-free ESC condition. n = 2 for all experiments in biological replicates.(G and H) Genome-wide transcriptional profiling followed by gene set enrichment analysis highlights that TBX3 selectively enriches mesoderm and endoderm (G) at the expense of trophoblast genes (H).(I) Principal component analysis (PCA) positions TBX3-induced cells (TBX3_d4+dox) close to endoderm.(J) Hierarchical clustering of TBX3-expressing EBs overlaps with published transcriptomes from DE (Christodoulou et al., 2011).∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S2.
	Figure 4. TBX3 Expression Promotes Mature Mesendodermal Cell Types(A–G and I) TBX3 contributes to both pancreatic, hepatic, and cardiovascular lineage specification as assessed by qPCR (A, C, E, F, and I) and IF (B, D, and G) at the indicated time points (pancreatic: Hnf1β, PDX1; hepatic: Hnf4α; ALB, albumin; cardiac: Myl2a, Islet1, Myl2v, Myh6, and α-ACTININ; vascular: CD31, VEGF-A, KDR). All scale bars are 20 μm. Nuclei in DAPI (blue).(H) Assessment of cardiac beating cluster percentage (y axis) and intensity (x axis) upon different Dox-treatment regimens. Error bars indicate standard error of the means (SEM). n = 3 for all experiments in biological replicates.∗p < 0.05; ∗∗p <0.01; ∗∗∗p < 0.001. See also Figures S4, S6, and S7 and Movies S1 and S2.
	Figure 5. TBX3 Activates a Nodal/Smad2 Signaling Cascade(A and B) Genome-wide transcriptional profiling to identify fluctuations in developmental signaling pathways in response to overexpression of TBX3 (+Dox). (A) Significant upregulation of the Nodal gene within the Dox-treated sample, whereas Wnt3a and Bmp4 are downregulated. (B) NODAL target genes Lefty1, Lefty2, and Cer1 are upregulated in the +Dox-treated sample.(C) qPCR analysis for Nodal mRNA in ±Dox cells at day 4 of differentiation. n = 3 in biological replicates.(D) Quantification of DNA fragment enrichment by ChIP using TBX3-specific antibody relative to control isotype measured by qPCR utilizing region specific Nodal primers (Figures S4A and S4B).(E and F) WB analysis for phosphorylated SMAD2 in day 4 EBs overexpressing TBX3 (+Dox) and control EBs (–Dox). TBX3-induced EBs contain higher levels of phosphorylated SMAD2 (F). n = 3 for biological replicates; quantification of three independent experiments is from (E).(G and H) TBX3 activates direct (G) and indirect (H) target genes of the NODAL/SMAD2 pathway. (G) A set of published genes showing direct (in absence of protein synthesis) or (H) indirect (in presence of protein synthesis) regulation upon SMAD2 phosphorylation was used for gene set enrichment analysis of ±Dox samples. Error bars = SEMs.(I) qPCR analysis for Eomes, Nodal, Hex, and FoxA2 mRNA levels in ±Dox cells at day 4 of differentiation with/without ALK4/5/7 inhibitor SB431542 (10 μM). n = 2 for independent experiments, each in biological triplicates.∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figures S3 and S4.
	Figure 6. TBX3 Acts via Non-Cell-Autonomous Mechanisms to Direct Cell Fate(A) Illustration of the treatment strategy to generate conditioned medium (CM) from iTBX3 cells and subsequent generation of EBs.(B) qPCR for Eomes, T, Foxa2, and Mesp1 in differentiating wild-type ESCs cultured in either non-CM or +Dox-CM days 0–4 from iTBX3 cells on day 4.(C–E) IF of differentiating wild-type ESCs cultured in either non-CM or CM from iTBX3 cells (either ±Dox) on day 4; wild-type cells treated with +Dox-CM days 0–4 of iTBX3 cells show a higher protein expression of the depicted markers (C) EOMES, (D) T, and (E) SOX17. All scale bars are 20 μm. Nuclei in DAPI (blue). n = 3 in biological replicates.(F) qPCR analysis for Nodal, Lefty1, and Lefty2 mRNA levels in either non-CM or +Dox-CM days 0–4 from iTBX3 cells on day 4.(G) Nodal, Eomes, T, and Sox17 mRNA levels in untreated (-Dox) and Dox-treated (+Dox) iTBX3 cells at day 4 of differentiation in ± 3 μM NODAL-blocking antibody, (n = 2) independent experiments, each in biological triplicates.∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3.
	Figure 7. TBX3 Directly Regulates Key Factors for Mesendoderm Induction(A) Luciferase reporter of EOMES (left bar), T (middle bar), and SOX17 (right bar) promoters were cotransfected in HEK cells with a TBX3-expression plasmid and assessed for luciferase activity, (n = 3) biological replicates. Similar data with iTBX3 ESCs (data not shown).(B–E) qPCR quantification of DNA fragments enrichment by ChIP using TBX3-specific antibody relative to control isotype for (B) Eomes, (C) T, (D) Sox17, and (E) Fgf2. n = 3 for biological replicates.(F and G) TBX3 induces OCT4 expression: (F) mRNA level of Oct4 upon TBX3 induction (±Dox) at indicated time points. (G) Protein level of OCT4 whole-mount stained EBs upon TBX3 induction (±Dox) at day 4. All scale bars 20 μm. Nuclei in DAPI (blue). n = 3 for biological replicates.(H) Schematic outline of OCT4 reporter assays (left panel). Luciferase reporter constructs with either proximal or distal OCT4 enhancer elements cotransfected into HEK cells together with a TBX3-expression plasmid and assessed for luciferase activity. n = 3 for biological replicates. Similar observations were made when iTbx3 ESCs were used (data not shown).(I) HEK cells were cotransfected with the respective luciferase reporter constructs in the presence or absence of OCT4- and TBX3-expression plasmids (schematic outline, left panel) and assayed for promoter activity of Sox17 (middle panel) and Eomes (right panel). Similar observations were made when iTbx3 ESCs were used (data not shown). n = 3 for biological replicates.∗p < 0.05; ∗∗p <0.01; ∗∗∗p < 0.001. See also Figures S4 and S7.

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