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. 2021 Jan 19;34(3):108655. doi: 10.1016/j.celrep.2020.108655

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© 2021 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Differential expression of E-cadherin and F-actin in polar trophectoderm and epiblast

(A) E-cadherin staining of embryos fixed upon implantation up to egg-cylinder formation. Fire staining represents intensity of signal, with purple indicating lowly expressed and yellow indicating highly expressed.

(B) F-actin staining of embryos fixed at consecutive stages from implantation to egg-cylinder formation; intensity is represented through fire staining, as in (A). A clear increase in the actin intensity from stage I to stage V is visible in the polar TE.

(C) pMyosin-II staining of embryos from implantation to egg-cylinder formation. Intensity is represented through fire staining, as in (A). pMyosin-II exhibits similar staining pattern as F-actin.

(D) Schematic illustration of the intensity measurements on tissue level (epiblast is indicated in magenta-purple, and green stripes indicate the area of tissue intensity measurement; polar TE is indicated in blue, and white stripes indicate the area of tissue intensity measurement) and level at which plot profiles were taken (red).

(E) Quantitative analysis of the E-cadherin intensity ratio. For each embryo, the mean gray value of both tissues was determined at 3 different z positions. The mean of the ratio polar TE/epiblast was plotted. As clearly visible, the polar TE intensity increased significantly over time. Scatterplot, mean ± SEM. Stage I, n = 35; stage II, n = 30; stage III, n = 19; stage IV, n = 24; stage V, n = 14. Analysis, unpaired Student’s t test: stages I–II, p = 0.0007; II–III, p = 0.0949; II–IV, p < 0.0001; IV–V, p = 0.5729.

(F) Quantitative analysis of the relative F-actin intensity (polar TE/epiblast) over time averaged for each embryo from measurements of the mean gray value of both tissues at 3 different stages. Scatterplot of average values with mean ± SEM. Relative actin intensity clearly increased over time. Stage I, n = 63; stage II, n = 65; stage III, n = 48; stage IV, n = 33; stage V, n = 32. Analysis, unpaired Student’s t test: stages I–V, p = 0.0100.

(G) Expression analysis of F-actin (green), pMyosin-II (red), and E-cadherin (blue) in the polar TE from stage I to stage III. White rectangles in the full figures illustrate the zoom-in region.

(H) Merged plot profiles of the apical surface of the polar TE in (G). A spline fit line was drawn with a thickness of 5 μm. Plot profile was determined through Fiji. Staining: F-actin (green), pMyosin-II (red), and E-cadherin (blue). It is visible that, from stages I–IV, the peaks of each marker begin to overlay.

(I) Representative immunofluorescent (IF) stainings of E4.5 embryos cultured for 20 h in hanging-drop culture supplemented with 100 μM Blebbistatin or DMSO in controls. Embryos were stained for F-actin (green), pMyosin-II (white), Cdx2 (red), and Gata6 (blue); the experiment was carried out 4 times. The shape of the EPIBLAST was annotated through a white dotted line.

(J) Quantitative analysis of the total curvature angle of the tissue interface epiblast/polar TE in treated embryos versus controls. Control, n = 12; treated, n = 11. Analysis, unpaired Student’s t test: p = 0.0249; treated and control embryos differ significantly.

Scale bars, 20 μm.