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

Figure 6. BMP signaling in micropatterns and embryos correlates with embryonic and extraembryonic mesoderm fates.

(A) Transverse cryosection of immunostained embryo in Figure 6—figure supplement 1B. Scale bar, 50 μm. (B) Quantification of pSMAD1/5/8 and BRACHYURY fluorescence intensity in E6.5 embryos. Cells within the epiblast, primitive streak (PS) and mesoderm were manually selected on confocal images of transverse cryosections in ImageJ as shown in right-hand panel. PS = BRACHYURY positive cells at embryo posterior. Mesoderm = cells positioned between VE and Epi. Quantification was carried out on three cryosections per embryo. N, number of cells. Data represents mean fluorescence intensity ± S.D. normalized to Hoechst fluorescence. (C) MIPs of immunostained colonies differentiated as in Figure 2E. Second panel depicts high magnification of colony edge. Scale bars, 100 μm. BRA, BRACHYURY; pS1/5/8, phosphorylated SMAD1/5/8. (D) Depiction of spatial patterning across multiple colonies. Each dot represents a single cell. (E) Quantification of voxel fluorescence intensity of pSMAD1/5/8 from colony center (0 μm) to edge (500 μm). Data represents average voxel intensity across multiple colonies. pSMAD1/5/8 colony numbers (n) in upper right corner. Data relative to maximum voxel intensity across the time course for each marker.

Figure 6.

Figure 6—figure supplement 1. BMP signaling is active in the posterior primitive streak, embryonic and extraembryonic mesoderm.

Figure 6—figure supplement 1.

(A) Schematic diagram depicting the sources of BMP (pink) within gastrulating embryos, described in (Lawson et al., 1999). At the start of gastrulation (E6.5-E6.75), BMP4 is expressed by the extraembryonic ectoderm and later (E7.5-E8.0) in the allantois, amnion and chorion. (B,D,E) Representative confocal images of immunostained gastrulating embryos showing BMP signaling activity based on nuclear localization of pSMAD1/5/8. Images represent maximum intensity projections (MIP), sagittal optical sections and transverse cryosections. Transverse section ‘b’ from panel B is shown in Figure 6A. Non-nuclear anti-BRACHYURY VE fluorescence represents to non-specific binding. AVE, anterior visceral endoderm; Epi, epiblast; ExE, extraembryonic ectoderm; VE, visceral endoderm; A-PS, anterior primitive streak; Al, allantois; M1, mesoderm 1; M2, mesoderm 2; A, anterior; P, posterior; Pr, proximal; D, distal; R, right; L, left; BRA, BRACHYURY; E-CAD, E-CADHERIN; pS1/5/8, pSMAD1/5/8. Scale bars, 50 μm. Dashed lines mark transverse plane. Dashed boxes outline regions in higher magnification in lower panels. In F, white bracket demarcates the posterior primitive streak and red bracket the anterior primitive streak. (C,G) Scatter plots showing the levels of pSMAD1/5/8 and BRACHYURY in arbitrary units (a.u.) within single mesoderm cells of early streak (E6.5-E6.75 - C) and early headfold (E7.75-E8.0 - G) embryos. Each dot represents a single cell. Linear regression curves were fitted to the points (red line). (F) Quantification of pSMAD1/5/8 and BRACHYURY fluorescence intensity in arbitrary units (a.u.) in early headfold (E7.75-E8.0) embryos. Cells within the epiblast, primitive streak (PS) and Mesoderm 1 and 2 (Meso1/2) were manually selected on confocal images of transverse cryosections using ImageJ software as shown in right-hand panel. The PS was identified as BRACHYURY-positive cells at the embryo posterior. Mesoderm cells were separated into two categories, those that had migrated further anteriorly and expressed low BRACHYURY and high pSMAD1/5/8 (Meso1) and those closest to the PS expressing high BRACHYURY (Meso2). Quantification was carried out on three cryosections per embryo. N, number of cells. Data represents mean fluorescence intensity normalized to the fluorescence intensity of Hoechst nuclear stain ± S.D. (H) Quantification of marker coexpression by voxel. Each dot indicates the fluorescence intensity of a single voxel, in arbitrary units (a.u.). Color represents voxel density within the plot. Gates were defined based on the 0 hr time point where BRACHYURY and CDX2 were not expressed and pSMAD1/5/8 was expressed only at low levels. Numbers within each quadrant represent the % of voxels within gate, rounded to the nearest whole number. N, number of colonies. (I) Pie charts illustrating the % of pSMAD1/5/8-positive cells that coexpress BRACHYURY or CDX2. Percentages shown within the largest fraction.
Figure 6—figure supplement 2. Bypassing the WNT receptor alters spatial patterning.

Figure 6—figure supplement 2.

(A) Simplified schematic of the Wnt pathway. In the absence of WNT receptor binding (left panel), GSKβ phosphorylates β-catenin, targeting it for degradation. β-catenin target genes are inactive. When WNT binds to the receptor (middle panel), factors downstream of the receptor are activated and inhibit GSKβ activity. β-catenin is not phosphorylated and not degraded, but instead translocates to the nucleus and activates target genes. CHIR99021 (CHIR) is a small molecule inhibitor of GSKβ. CHIR directly inactivates GSKβ independent of WNT receptor activation (right panel). As with WNT receptor binding, inactive GSKβ cannot phosphorylate β-catenin hence target genes are activated. (B) Schematic diagram of differentiation conditions. EpiLCs were generated as described in Figure 1C. EpiLCs were plated overnight onto Laminin-coated micropatterns (−24 hr) in N2B27 medium with 12 ng/ml FGF2 and 20 ng/ml ACTIVIN A (F/A). Various conditions were then used for further differentiation, F/A with BMP4 (50 ng/ml) and WNT3A (200 ng/ml) (Control) or F/A, BMP4 and 3 μM CHIR99021, a small molecule inhibitor of GSK3. Colonies were analyzed after 72 hr differentiation. (C,E) Confocal maximum intensity projections of immunostained colonies after 72 hr of differentiation. Scale bars, 100 μm. (D,F) Quantification of immunostaining voxel fluorescence intensity, in arbitrary units (a.u.), from colony center (0) to edge (500). Data represents average voxel intensity across multiple colonies.