Figure 4—figure supplement 2

(A) High-resolution fluorescence images of 50% epiboly control and dorsal determinants removed (DD-removed) embryos stained both for 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). The proportion of embryos with a phenotype similar to the images shown is indicated in the lower left corner (control: n = 24, N = 8; DD-removed: n = 25, N = 7). (B) Percentage of control (n = 24, N = 8) and DD-removed (n = 25, N = 7) embryos showing a domain of pSMAD2/3 positive nuclei (present), a few sporadic pSMAD2/3 positive nuclei (strongly reduced) or no positive nuclei (negative) at 50% epiboly (see Materials and methods for additional details). (C) Percentage of pSMAD2/3 positive nuclei in control (n = 10, N = 3) and DD-removed (n = 9, N = 3) embryos at 50% epiboly. ns, not significant (Unpaired t test). (D) Normalized intensity of the brightest pSMAD2/3 nuclei (for details see Materials and methods) in control (n = 10, N = 3) and DD-removed (n = 9, N = 3) embryos at 50% epiboly. ns, not significant (Mann-Whitney test). (E) High-resolution images of sebox::EGFP-expressing control (n = 20, N = 2) and DD-removed (n = 29, N = 2) embryos at bud stage. The embryos are shown as side views and the proportion of embryos expressing sebox::EGFP shown is indicated in the lower right corner. (F) Percentage of control (n = 20, N = 2) and DD-removed (n = 29, N = 2) embryos showing normal, reduced or no sebox::EGFP expression at bud stage (see Materials and methods for additional details). (G) High-resolution fluorescence images of 50% epiboly control (n = 18, N = 4) and CA-Alk8 (30 pg; n = 10, N = 4) overexpressing blastoderm explants stained for both pSMAD2/3 (pink) and DAPI (grey). Nuclear pSMAD2/3 is shown in a fire lookup table 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. The proportion of blastoderm explants with a phenotype similar to the images shown is indicated in the lower left or right corner. (H) Percentage of control (n = 18, N = 4) and CA-Alk8-overexpressing (30 pg; n = 10, N = 4) blastoderm explants showing a domain of pSMAD2/3 positive nuclei (present), a few sporadic pSMAD2/3 positive nuclei (strongly reduced) or no positive nuclei (negative) at 50% epiboly (see Materials and methods for additional details). Scale bars: 100 µm (A,E,G).

Figure 4—figure supplement 2—source data 1.