Fig 2: Positive Feedback Promotes Switch-like, Irreversible Activation of the SMAD Regulatory Network and Sustains BMP4 Signals(A) Top: representative images of SMAD activation dynamics after BMP4 (50 ng/mL) treatment. Scale bar represents 100 µm. Bottom: SMAD activation dynamics in space after BMP4 treatment, assuming a circular geometry for hESC colonies.(B) Quantification of SMAD activation after BMP4 (50 ng/mL) treatment. More than 200 cells were analyzed for each experimental condition. n = 3 independent experiments.(C) Quantification of SMAD activation dynamics as a function of BMP4 concentration.(D) Percentage of cells showing active (red) and inactive (blue) SMAD as a function of BMP4 concentration. Error bars represent means ± standard deviations (SDs).(E) Mean intensity of fitted Gaussian distributions corresponding to cells with active (red) and inactive (blue) SMAD for a range of BMP4 concentrations. The dotted line highlights the BMP4 concentration at which two populations of active and inactive SMAD are seen. More than 200 cells were analyzed for each experimental condition. n = 3 independent experiments.(F) Quantification of SMAD activity at steady state (60 min) in response to increasing concentrations of Noggin. Peak levels of active SMAD were normalized to 1. Basal activity at time 0 is shown. n > 200 cells were analyzed for each experimental condition. n = 3 independent experiments. Error bars represent means ± standard deviations (SDs).(G) Quantification of SMAD activity at steady state (60 min) in response to increasing concentrations of BMP receptor inhibitor (K02288). Peak levels of active SMAD were normalized to 1. Basal activity at time 0 is shown. n > 200 cells were analyzed for each experimental condition. n = 3 independent experiments. Error bars represent means ± standard deviations (SDs)(H) Top: schematic showing time of BMP4 and Noggin stimulation for each experimental condition. Bottom: representative images of SMAD activation after BMP4 stimulation followed by Noggin (100 ng/mL) treatment before or after SMAD full activation. Cells cultured with either BMP4 or Noggin alone were used as positive and negative controls, respectively. Scale bars represent 100 µm. n > 200 cells were analyzed for each experimental condition.(I) Heatmap showing a subset of RNA-seq-based gene expression profiles showing graded expression dynamics after BMP4 stimulation. BMP type I and type II receptors are highlighted.(J) Quantification of BMP receptor (BMPR1A) expression after BMP4 stimulation in the presence (blue) or absence (red) of Noggin (100 ng/mL) as measured by qPCR. Housekeeping gene GUSB was used for normalization. Error bars represent ± SDs. n = 3 independent experiments.(K) Quantification of BMP receptor (BMPR1A) expression in the presence or absence of BMP4 (50 ng/mL), as measured by mRNA-FISH. Error bars represent ±SDs. n > 200 cells were analyzed for each experimental condition (****p < 0.01).(L) SMAD1 ChIP-seq analysis of intron 2 of BMPR1A in the presence (red) or absence (blue) of BMP4 showing positive feedback between SMAD and its activator, BMPR1A. Significant peak region relative to input chromatin is highlighted. The amplicon used for ChIP qPCR (qPCR_Int2) is shown.(M) Quantification of SMAD1 ChIP-qPCR using qPCR_Int2 as an amplicon in the presence (red) or absence (blue) of BMP4 (50 ng/mL). A primer set -3 kb from BMPR1A was used as a negative control. Error bars represent means ± SDs for n = 3 biological replicates (**p < 0.01, ns is not significant).

Fig 3: Antibody 3F6 has no effect on BMP-responsiveness in BMPR2 knock-down HEK293T cells. a Expression levels of endogenous BMPR2 in scramble control HEK293T cells (Control) and HEK293T cells carrying anti-BMPR2 shRNA (BMPR2-KD) compared to HPRT1 loading control. Data are expressed as normalized to scramble control (Control) using the 2−∆∆Ct method. n = 6 per condition. Asterisk indicates p < 0.05 by unpaired t test. b, c Expression of endogenous BMPR2 in scramble control HEK293T cells (Control) and BMPR2-KD HEK293T cells compared to β-actin loading control. Approximate molecular weights are indicated in b. Representative immunoblot is shown in b and results from three independent runs are quantified in c (data are expressed as mean ± SEM ratio of BMPR2: β-actin normalized to scramble control (Relative Expression)). Asterisk indicates p < 0.05 by paired t test. d, e BMP2 induces phosphorylation of SMAD1, 5, and 8 (pSMAD1,5,8) in BMPR2-KD HEK293T cells. No effect on BMP2-responsiveness of BMPR2-KD HEK293T cells was observed with pre-treatment using control ascites (Ctrl Asc.) or 3F6. Approximate molecular weights are indicated in d. Results are quantified in E and expressed as mean ± SEM ratio of phosphorylated SMAD1,5,8: total SMAD1 relative to BMP2 treatment alone (Relative pS1:S1). n = 4 per condition

Fig 4: Antibody 3F6 reduces activation of BMP pathway in HEK293T cells. a Expression of endogenous BMPR2 by HEK293T cells compared to β-actin loading control. Approximate molecular weights are indicated. b, c BMP2 induces phosphorylation of SMAD1, 5, and 8 (pSMAD1,5,8) in HEK293T cells and this response is blunted by pre-treatment with 3F6. Approximate molecular weights are indicated in b. Results are quantified in c and expressed as mean ± SEM ratio of phosphorylated SMAD1,5,8: total SMAD1 relative to BMP2 treatment alone. No effect on BMP2-responsiveness was observed with pre-treatment using control ascites (Ctrl Asc.). n = 3 per condition. Asterisk indicates p < 0.05 by paired t test

Fig 5: BMP signaling is repressed in Ddx6△/△ pluripotent cells.(A) Cell counting of ESCs over a three-day culture period. Mean ± SEM. Significance was calculated by the Student’s t-test (n = 5). (B) qRT-PCR examining the relative expression of pluripotency markers in Ddx6△/△ ESCs to WT ESCs. Mean ± SEM. Student’s t-test (n = 7~9). (C) Cell counting during the ESC-to-EpiLC induction period. Mean ± SEM. Student’s t-test (n = 13 for WT, n = 7 for Ddx6△/△). (D) qRT-qPCR examining the expression pattern of Nodal, Fgf5, and Zic3. Mean ± SEM. Student’s t-test (n = 9 for Nodal & Fgf5, n = 7 for Zic3) (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (E-F, I) qRT-PCR analysis of the expression trend of several key genes during the EpiLC induction period. Each bar represents the relative expression of Ddx6△/△ cells to WT cells at the indicated time point. Mean ± SEM. Student’s t-test. (E) Major pluripotency genes (n = 7~9). (F) Early neuroectoderm and mesendoderm lineage markers (n = 7~9). (I) The negative regulators of BMP signaling (n = 6~9). (G) TuJ1 ICC (immunocytochemistry) on Day1 of monolayer differentiation (Scale: 50 μm) (n = 3). (H) qRT-PCR analysis of the expression of the known BMP-SMAD1/5 target genes in ESCs. Each value is the relative expression to WT ESCs (n = 5). (J) Western blot for endogenous SMAD5 and phosphorylated SMAD1/5 in mESCs. Four conditions: LDN (LDN193189: BMP Type I receptor ALK2/3 inhibitor)/—(non-treated)/ BMP4 1/10 (1/10 dilution of rBMP4 treated sample)/ BMP4 (rBMP4 treated). The quantified signal intensity of the band is displayed on the right-side graph (n = 6).