Fig 2: P300 Levels Tune Response to LIF and BMP4 and Adjust LIF Responsiveness and Conversion Frequencies(A) QRT-PCR on Lif-r, Gp130, and Stat3 after siRNA knockdown with scramble control (Neg.) or a pool of siRNAs specific for P300 (P300i) on (i) non-µP CDEs and (ii) µP CDEs. Transcript levels normalized to siRNA-free CDEs.(B) LIF responsiveness in EpiSCs was assayed in the presence of LIF and/or BMP after siRNA knockdown of P300 (P300i). Scrambled siRNA was used as the control (Neg.)(C) (i) Scatterplots of P300i cells versus Neg. cells after treatment with nothing (AF) or LIF and BMP4 followed by a 15 min pulse of LIF to assay for LIF responsiveness. (ii) Representative images of P300i and Neg. cells treated with LIF and BMP4 prior to pulsing with LIF. Cells were costained for pSTAT3 (red) and P300 (green).(D) ALP colony counts (insets) of knockdown cells in 2iL media following treatment in nothing (AF), LIF, BMP4, or LIF + BMP4.(E) Schematic of model. (i) Briefly, EpiSCs residing in an area of low local cell density secrete low levels of endogenous GP130 ligands and BMP4. (ii) In regions of high local cell density, whether by µP, MEFs, or in high-confluence culture, endogenous GP130 ligands and BMP4 accumulate to regulate transcription of Lif-r through binding to P300. Endogenous signaling may be recapitulated by addition of LIF and BMP4, and results in increased LIF responsiveness and subsequent increased conversion frequency.Error bars represent SDs n = 3 for (A) and n = 4 for (B) (both technical replicates, independent wells) all images are representative.

Fig 3: Administration of Zeb1?EC mice with r.Dll4 protein efficiently restores the impaired bone angiogenesis and osteogenesis.a, b Control and ZEB1-deleted bone ECs seeded on vehicle- or r.Dll4-precoated chamber wells (or culture dishes) were subjected to NICD1 immunostaining (a) and immunoblotting (b) analyses, respectively. Representative confocal or immunoblot images as shown are from three independent experiments. Scale bar, 20 µm. c Representative confocal images of CD31/EMCN and VEGFA immunostaining in the tibia of Zeb1WT and Zeb1?EC mice that were i.p. injected with 1 µg/g r.Dll4 protein at P4 for 2 consecutive weeks before analysis at P21 (n = 5, each). Scale bar, 100 µm. d Quantification of type H vessel density (top panel) and VEGFA staining levels (bottom) in the tibia as shown in c (n = 5 independent experiments). e Micro-CT analysis of the tibia showing improved bone formation in trabecular bone (top panels) and cortical bone (bottom panels) of Zeb1?EC mice treated with r.Dll4 protein as described in c (n = 5, each). Scale bar, 0.2 mm. f Quantification of bone architectures in the tibia as shown in e (n = 5 independent experiments). g Runx2, Osterix, and ALP immunostaining of the tibia as described in c (n = 5, each). Scale bar, 100 µm. h Quantification of Runx2+, Osterix+, and ALP+ cells in the tibia as shown in g (n = 5 independent experiments). All data are represented as mean ± SD. **P < 0.01. Differences are tested using one-way ANOVA with Tukey’s post hoc test (d, f, h). The source data are provided as a Source Data file. Unprocessed original scans of blots are shown in Source Data file.

Fig 4: LIF and BMP4 Increase LIF Responsiveness in EpiSCs On and Off µP(A) Immunostaining for pSTAT3 and OCT4 in (i) CDEs and (ii) embryo-derived EpiB3 treated with LIF and/or BMP4 then pulsed with LIF to assay LIF responsiveness. (iii) Quantification of images seen in (i) and (ii).(B) Immunostaining for pSTAT3 and OCT4 after inhibition of BMP signaling on µP by (i) NOGGIN (Nog. 300 ng/ml), (ii) LDN-193189 (LDN, 0.1 µM), or (iii) siRNA against Smad1 (Smad1i) followed by a LIF pulse to assay LIF responsiveness.(C) ALP staining of revertant CDEs after treatment with any combination of LIF and BMP4 (both at 10 ng/ml). Quantification on bottom panel, *p < 0.05, as tested by a two-sided, two-sample t test.All data are presented as mean ± SD (A: n = 4 technical replicates, independent wells; B: n = 6 for Bi, n = 4 for Bii, all technical replicates, independent wells; C: n = 3, biological replicates across independent experiments).

Fig 5: EC-specific deletion of ZEB1 impairs angiogenesis-dependent bone formation.a Representative images of freshly dissected tibia of 3-week-old Zeb1WT and Zeb1?EC mice (n = 5, each). Scale bar, 50 mm. b Representative micro-CT images of trabecular bone (top panels) and cortical bone (bottom panels) of 3-week-old Zeb1WT and Zeb1?EC mice (n = 5, each). Scale bar, 0.2 mm. c Quantification of Tb. BV/TV trabecular separation (Tb. Sp.), Tb. Nb. trabecular thickness (Tb. Th.), cortical thickness (Ct. Th.) in the tibia as shown in b (n = 5 independent experiments). d Representative images of calcein double labeling of Zeb1WT and Zeb1?EC tibia (n = 5, each). Scale bar, 100 µm. e Quantification of dynamic bone formation in tibia as shown in d (n = 5 independent experiments). f, h, j Representative images of 3-week-old Zeb1WT and Zeb1?EC tibia (n = 5, each) immunostained with Runx2 (f), Osterix (h), and ALP (j). Scale bar, 100 µm. g, i, k Quantification of Runx2+ (g), Osterix+ (i), and ALP+ (k) cells in the tibia as shown in f, h, j (n = 5 independent experiments). l Representative images for Alizarin red S staining of 3-week-old Zeb1WT and Zeb1?EC tibia (n = 5, each). Scale bar, 100 µm. All data are represented as mean ± SD. **P < 0.01; NS not significant. Differences are tested using unpaired two-tailed Student’s t-test. The source data are provided as a Source Data file.