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. 2021 Sep 27;10:e65068. doi: 10.7554/eLife.65068

Figure 3. GATA3 expression is initiated independently of cell polarization.

(a) Representative images of in vitro fertilized human embryos warmed at day 3 and cultured for 2, 9, or 24 hr (see scheme in Figure 1a ) to reveal the localization of F-actin, PARD6, and GATA3. White arrowheads indicate outer cells; yellow arrowheads indicate inner cells. (b) Quantification of the number of GATA3-positive cells in embryos from panel a. Cells that have higher nuclear GATA3 signal (above a nucleus to cytoplasm ratio of 1.5) are categorized as GATA3+ cells. Numbers in each bar indicate the number of cells analyzed. ns, not significant, Fisher’s exact test. N = 17 embryos for 2 hr N = 20 embryos for 9 hr and N = 33 embryos for 24 hr. (c) Quantification of GATA3 nuclear signal in embryos from panel a. Each dot represents one cell. Data are shown as mean ± S.E.M. The GATA3 nuclear signal has been calculated as the nucleus to cytoplasmic signal ratio in each cell. N = 21 cells (2 hr), N = 32 cells (9 hr), and N = 49 cells (24 hr). **p < 0.01; two-tailed unpaired Student’s t-test. (d) Representative images of embryos injected with Control siRNA or PLCB1+ PLCE1 siRNA at the zygote stage, cultured until Day 4, and stained for F-actin, PARD6, and GATA3. (e), Quantification of GATA3 nuclear signal intensity (normalized against the cytoplasmic signal) in embryos from panel d. Each dot indicates one cell analyzed. N = 31 cells for Control siRNA group and N = 44 cells for PLCB1+ PLCE1 siRNA group. ****p < 0.0001, Mann-Whitney test. Five independent experiments (a–c) and two independent experiments (d-f). Scale bars, 15 µm. (f), Quantification of the level of nuclear GATA3 (normalized against cytoplasm signal) in embryos injected with Control siRNA or PLCB1+ PLCE1 siRNA. The data were classified according to the polarity status of the cells. Each dot indicates one cell. N = 31 cells for Control siRNA and N = 44 cells for PLCB1+ PLCE1 siRNA, ****p < 0.0001, Mann-Whitney test.

Figure 3—source data 1. Source data for Figure 3 and Figure 3—figure supplement 1.
This file contains the source data used to make the graphs presented in Figure 3. GraphPad Prism was utilized to visually represent the quantitative data.

Figure 3.

Figure 3—figure supplement 1. Trophectoderm specification and inner cell generation in human embryos.

Figure 3—figure supplement 1.

(a) Number of cells showing nuclear GATA3 (defined as a nucleus to cytoplasm ratio of more than 1.5) in relation to their polarity status in human embryos at embryonic day 4. Numbers in each bar indicate the number of cells analyzed. N = 13 embryos. (b) Human embryos at embryonic day 4 immunostained for YAP1, PARD6, and DAPI. White arrows indicate outer cells, yellow arrows indicate inner cells. (c) Quantification of the nucleus to cytoplasm ratio of YAP1 in embryos from panel b. Each dot represents a cell. N = 22 polarized cells and N = 18 unpolarized cells from four embryos. ****p < 0.0001, Mann-Whitney test. (d) Representative images of embryos treated with different concentrations of U73122, cultured from day 3 to day 4 and stained for F-actin, PARD6, and GATA3. (e) Quantification of GATA3 nuclear signal intensity (normalized against the cytoplasmic signal) in embryos from panel d. N = 142 cells for Media control; N = 37 cells for DMSO 35 mM; N = 84 cells for DMSO 53 mM, N = 56 cells for U73122 5 µM (35 mM DMSO) and N = 90 for U73122 7.5 µM (53 mM DMSO). Each dot represents one cell analyzed. ns: non-significant. **** p < 0.0001, Kruskal-Wallis test with a multiple comparisons test. Individual paired comparisons: ****p < 0.0001 (Media control vs. U73122 7.5 µM, DMSO 53 mM vs. U73122 7.5 µM, and U73122 5 µM vs. U73122 7.5 µM), and *p = 0.0227 (Mediacontrol vs. DMSO 35 mM). The rest of the comparisons were not statistically significant. Scale bars, 15 µm.