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Figure 2 (A) High-resolution fluorescence images of stage-matched embryos (dorsal view) and blastoderm explants (top view) at 50% epiboly, germ ring and shield stage stained for both pSMAD2/3 (pink) and DAPI (grey). Nuclear pSMAD2/3 is color-coded using a fire lookup table (highest intensities in yellow) and was masked based on the DAPI signal. Insets are zoom-in images of the highlighted regions (dashed boxes). Yellow circles denote the wounding site in explants. The proportion of embryos and explants with a phenotype similar to the images shown is indicated in the lower left corner (50% epiboly: embryos, n = 12, N = 4, explants, n = 21, N = 4; germ ring: embryos, n = 16, N = 4, explants, n = 25, N = 4 and shield: embryos, n = 6, N = 3, explants, n = 10, N = 3). (B) Percentage of pSMAD2/3 positive nuclei in stage-matched embryos and blastoderm explants at 50% epiboly (embryos: n = 7, N = 3; explants: n = 10, N = 3), germ ring (embryos: n = 10, N = 4; explants: n = 10, N = 4) and shield stage (embryos: n = 6, N = 3; explants: n = 8, N = 3). ****p<0.0001, ns, not significant (ANOVA test). (C) Normalized intensity of the brightest pSMAD2/3 nuclei (for details see Materials and methods) in stage-matched embryos and blastoderm explants at 50% epiboly (embryos: n = 7, N = 3; explants: n = 10, N = 3), germ ring (embryos: n = 10, N = 4; explants: n = 10, N = 4) and shield stage (embryos: n = 6, N = 3; explants: n = 8, N = 3). ****p<0.0001 (Kruskal-Wallis test). (D) Normalized distance, expressed as cell tiers, of the brightest pSMAD2/3 nuclei (for details see Materials and methods) from the YSL or wounding site in stage-matched embryos and blastoderm explants at 50% epiboly (embryos: n = 7, N = 3; explants: n = 10, N = 3), germ ring (embryos: n = 10, N = 4; explants: n = 10, N = 4) and shield stage (embryos: n = 6, N = 3; explants: n = 8, N = 3). ****p<0.0001 (Kruskal-Wallis test). (E) Bright-field single-plane images of bud stage MZoep (n = 40, N = 3), DMSO (treated from 256 c to bud, n = 81, N = 5) or Nodal inhibitor (SB-505124)-treated blastoderm explants (treated from 256 c to bud, n = 49, N = 4; 256 c to shield, n = 38, N = 3; shield to bud, n = 43, N = 4). (F) Percentage of extended or not-extended wildtype (n = 26, N = 3), MZoep (n = 40, N = 3), DMSO (treated from 256 c to bud, n = 81, N = 5) and Nodal inhibitor (SB-505124)-treated blastoderm explants (treated from 256 c to bud, n = 49, N = 4; 256 c to shield, n = 38, N = 3; shield to bud, n = 43, N = 4) at bud stage. (G) Circularity of bud stage wildtype (n = 26, N = 3), MZoep (n = 40, N = 3), DMSO (treated from 256 c to bud, n = 81, N = 5) and Nodal inhibitor (SB-505124)-treated blastoderm explants (treated from 256 c to bud, n = 49, N = 4; 256 c to shield, n = 38, N = 3; shield to bud, n = 43, N = 4). ****p<0.0001, *p=0.0112 (Kruskal-Wallis test). (H) Normalized extension length of extended DMSO (treated from 256 c to bud, n = 77, N = 5) and Nodal inhibitor (SB-505124)-treated blastoderm explants (treated from shield to bud, n = 33, N = 4) at bud stage. ns, not significant (Unpaired t test). Scale bars: 100 µm (A), 200 µm (E).