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. 2018 Feb 7;7:e32839. doi: 10.7554/eLife.32839

Figure 4. EMT is associated with micropatterned EpiLC differentiation.

Data from colonies differentiated as in Figure 2E. (A,B) Sagittal (A) and transverse sections (B) of late streak embryo. Dashed box marks high magnification region in lower panel. Dashed lines mark transverse planes in B. Non-nuclear anti-BRACHYURY VE fluorescence represents non-specific binding. A, anterior; P, posterior; Pr, proximal; D, distal; L, left; R, right; VE/DE, visceral endoderm/definitive endoderm; ExE, extraembryonic ectoderm; ExM, extraembryonic mesoderm; Epi, epiblast; Meso, mesoderm. Scale bars, 50 μm. (C) Quantification of colony height from colony center (0) to edge (500) across multiple colonies, three independent experiments, 0 hr: n = 11, 24 hr: n = 15, 48 hr: n = 17, 72 hr: n = 18. (D) Time-course showing brightfield images (upper panels) and MIPs of comparable immunostained colonies (lower panels). Scale bars, 100 μm. (E–G) Images of z-axis profile from colony center (left) to edge (right). (G–I) EpiLCs were plated onto micropatterns overnight with F/A. The following day medium was changed to F/A, BMP4, WNT3A (E,F) or medium blocking Activin/Nodal signaling - FGF2, BMP4, WNT3A, SB431542 (ACTIVINi, (G–I). (H) brightfield image of ACTIVINi colony. (I) MIPs of immunostained ACTIVINi colonies at 72 hr differentiation. Scale bars, 100 μm. BRA, BRACHYURY.

Figure 4.

Figure 4—figure supplement 1. Cells undergo an epithelial to mesenchymal transition during gastrulation and in vitro differentiation.

Figure 4—figure supplement 1.

(A) Quantification of immunostaining voxel fluorescence intensity (in arbitrary units, a.u.) of SOX2, BRACHYURY and CDX2 from colony center (0) to edge (500) as well as relative colony height. Data represents average voxel intensity or change in colony height across multiple colonies relative to the maximum value for each marker. (B) Confocal optical sections of the outer colony edge. Scale bars, 100 μm. (C) MIPs of immunostained 72 hr colonies. Scale bars, 100 μm. (D) Images of z-axis profile from colony center (left) to edge (right). (E) Quantification of immunostaining. Voxel fluorescence intensity was measured from colony center (0) to edge (500). Data represents average voxel intensity across multiple colonies (n = 9) relative to maximum voxel intensity for each marker. (F) EpiLCs were plated onto micropatterns overnight in N2B27 with 12 ng/ml FGF2 and 20 ng/ml ACTIVIN A (F/A). The following day medium was changed to 12 ng/ml FGF2, 50 ng/ml BMP4, 200 ng/ml WNT3A and 10 μM SB431542 (ACTIVINi). Confocal maximum intensity projections of immunostained colonies after 72 hr of differentiation. Scale bars, 100 μm. BRA, BRACHYURY.