Figure 2. Micropatterned EpiLCs undergo spatially organized differentiation.

(A) Maximum intensity projection (MIP), sagittal and transverse sections of an embryonic day (E) 6.5 mouse embryo. Dashed line marks transverse plane. Non-nuclear anti-BRACHYURY/CDX2/SOX2 VE fluorescence likely represents non-specific binding. ExM, extraembryonic mesoderm; PS, primitive streak; A, anterior; P, posterior; Pr, proximal; D, distal. Scale bars, 50 μm. (B) Lookup table (LUT) of SOX2 marking anterior Epi (A-Epi) and NANOG marking posterior Epi (P-Epi). Orange dashed lines delineate regions of interest. (C) Quantification (5 sections/embryo/ stage) of SOX2 and NANOG in manually selected (panel B) anterior (A) and posterior (P) Epi of E6.5-E6.75 and E7.0-E7.5 embryos, normalized to Hoechst fluorescence. Data depicts mean fluorescence intensity ± S.D. N, number of embryos. No NANOG was observed in the A-Epi hence ~0.5 a.u. equates to background signal. (D) BMP, Wnt, Nodal, FGF signaling initiates gastrulation at the P-Epi - extraembryonic ectoderm (ExE) boundary. BMP4 produced by ExE stimulates Wnt3 expression within proximal Epi. WNT3 produced by Epi and visceral endoderm (VE) triggers Nodal and Fgf8 expression. NODAL promotes Bmp4 expression in the ExE. The anterior VE (AVE) expresses Wnt and Nodal pathway antagonists, restricting signaling activity to P-Epi. (E) EpiLCs were plated onto Laminin-coated micropatterns overnight (−24 hr) in N2B27 with F/A. The following day medium was changed to F/A, BMP4, WNT3A for 72 hr. Colonies were analyzed at 24 hr intervals. (F) MIPs of immunostained 1000 μm diameter colonies. All subsequent data represents 1000 μm diameter colonies. Upper two panels represent a merge of the markers shown below. Second panel shows high magnification of colony edge. Scale bars, 100 μm. BRA, BRACHYURY. (G) Depiction of average positional marker expression across multiple colonies. Each dot represents a single cell. (H) Quantification of voxel fluorescence intensity from colony center (0) to edge (500). Data represents average voxel intensity relative to maximum voxel intensity across time course/marker. For 0,24,48,72 hr respectively, POU5F1/NANOG n = 5,3,3,3, SOX2 n = 15,7,21,20, BRACHYURY n = 11,9,10,12, GATA6/SOX17/CDX2 n = 3,5,6,5. Markers grouped by spatial distribution within colonies. OTX2 and FOXF1 only analyzed at 72 hr.

Figure 2—figure supplement 1. FGF, ACTIVIN and endogenous WNT induce a primitive streak state.

(A). Confocal sagittal optical section of an early streak embryo. Non-nuclear anti-BRACHYURY VE fluorescence likely represents non-specific binding. PS, primitive streak; A, anterior; P, posterior; Pr, proximal; D, distal. Scale bars, 50 μm. (B) EpiLCs were generated as in Figure 1C. EpiLCs were plated overnight onto Laminin-coated micropatterned surfaces (−24 hr) in N2B27 medium with 12 ng/ml FGF2 and 20 ng/ml ACTIVIN A (F/A). Cells were cultured for a further 72 hr in either N2B27 with F/A or F/A with 10 μM of XAV939 (Wnt signaling inhibitor, WNTi). (C) Representative confocal maximum intensity projections of immunostained 1000 μm diameter colonies after 72 hr in the conditions in panel D. Scale bars, 100 μm. (D) Quantification of SOX2 and BRACHYURY immunostaining voxel fluorescence intensity, in arbitrary units (a.u.), from colony center (0) to edge (500). Data represents average voxel intensity across multiple colonies. Control: n = 32, WNTi: n = 7. (E) Schematic diagram depicting cell fates generated after 72 hr of in vitro micropattern differentiation with FGF2 and ACTIVIN A (+XAV) or FGF2, ACTIVIN A and endogenous Wnt signaling and corresponding in vivo cell types.

Figure 2—figure supplement 2. Robust micropattern differentiation of EpiLCs.

(A) Confocal maximum intensity projection (MIP) showing micropatterned colonies of 1000, 500, 250, 140 and 80 μm diameter. Scale bar, 1000 μm. (B) Left panels show confocal MIP images of 5 independent 1000 μm diameter colonies (C1-5). Middle panels show quantification of SOX2, BRACHYURY and CDX2 immunostaining voxel fluorescence intensity, in arbitrary units (a.u.), from colony center (0) to edge (500). Data relative to maximum voxel intensity for each marker. Right panel shows average radial profile of these five colonies. (C) While much of the work in this study utilized E14 ESCs, comparable patterning was observed with other mouse ESC lines. Quantification of immunostaining voxel fluorescence intensity of differentiated T^GFP/+ and Sox17^GFP/+ cell lines, in arbitrary units (a.u.), from colony center (0) to edge (500). Data represents average voxel intensity across multiple colonies relative to maximum voxel intensity for each marker. For T^GFP/+ cells, GATA6, SOX2: n = 10, BRACHYURY, CDX2, T^GFP: n = 11. For Sox17^GFP/+ cells, SOX2: n = 5, BRACHYURY, CDX2, Sox17^GFP: n = 6, GATA6: n = 5. (D) Confocal MIPs of immunostained colonies differentiated as in Figure 2E. Scale bars, 100 μm. (E) High magnification confocal image of the colony edge after 24 hr in conditions defined in Figure 2E. Yellow arrowheads mark cells coexpressing BRACHYURY and SOX2.