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. 2024 Feb 12;151(3):dev202098. doi: 10.1242/dev.202098

Fig. 3.

Fig. 3.

Notch-FGF signalling crosstalk in hESC-derived NMPs. (A) Scheme of treatments during the differentiation of hESCs toward NMPs. (B) qPCR expression analysis of the indicated Wnt, FGF and Notch signalling pathway components in DAPT/DMSO-treated hESC-derived NMP cultures. Error bars represent mean±s.e.m. (n=3 independent experiments). **P≤0.01 (one-sample t-test and Wilcoxon test). (C,D) Representative western blot analysis of phospho-MAPK (p42/p44) in NOTCHi/DMSO-treated NMPs (C) and corresponding quantification (D). Error bars represent mean±s.e.m. (n=3 independent experiments). **P≤0.01 (paired two-tailed t-test). MGV, mean grey value; ns, not significant. (E) qPCR expression analysis of NMP markers in NOTCHi NMPs generated from hESCs using the standard (20 ng/ml) or high (40 ng/ml) FGF2 concentration. Error bars represent mean±s.e.m. (n=6 independent experiments). *P≤0.05, ***P≤0.001 (one-sample t-test and Wilcoxon test). (F) Image analysis of the percentage of nuclei positive for TBXT, HOXC9 and SOX2 protein expression. Graph shows mean values (n=3 independent experiments). NOTCHi data are taken from Fig. S2B. (G) qPCR expression analysis of the indicated HOX genes in NOTCHi NMPs generated from hESCs using the standard (20 ng/ml) or high (40 ng/ml) FGF2 concentration. Error bars represent mean±s.e.m. (n=3 independent experiments) *P≤0.05, **P≤0.01 (one-sample t-test and Wilcoxon test). (H) qPCR expression analysis of indicated pro-neural/mesodermal NMP and spinal cord neurectoderm markers in PD03-treated (FGFi) hESC-derived NMPs versus controls. Error bars represent mean±s.e.m. (n=3 independent experiments). *P≤0.05, **P≤0.01 (paired, two-tailed t-test). (I) qPCR expression analysis of indicated Wnt, FGF and Notch signalling pathway components in PD03-treated/control hESC-derived NMP cultures. Error bars represent mean±s.e.m. (n=3 independent experiments). *P≤0.05, **P≤0.01 ***P≤0.001 (one-sample t-test and Wilcoxon test). (J) Model depicting the crosstalk between Notch and FGF signalling in regulating HOX/proneural gene and TBXT expression in TBXT+/SOX2+ NMPs.