Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos

Abstract

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par-aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE-specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16-cell stage embryos. Similar to 32-cell stage embryos, disruption of the Par-aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32-cell stage embryos, 16-cell stage embryos with a disrupted Par-aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p-ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16-cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8-cell stage embryos form polar-apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16-cell stage is regulated by both Par-aPKC-dependent and -independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling.

Keywords: Hippo signaling; Par-aPKC; asymmetric cell division; cell polarity; preimplantation embryo.

Figure 1

Correlation between cell polarity and nuclear Yap levels during preimplantation development. (A) Distribution of an apical marker, phosphorylated ERM (p‐ERM), and Yap between the 4‐cell and blastocyst stages. 8c‐cell, compacted 8‐cell stage. (B) Changes in the ratio of nuclear to cytoplasmic Yap (Yap N/C ratio) in the outer cells (black) and the inner cells (red). An N/C ratio of 1 indicates that the intensity of the Yap signal in the nucleus and cytoplasm is the same. When the N/C ratio is >1, Yap is predominantly nuclear. Graphs show the mean ± SEM (4‐cell, n = 20; 8‐cell, n = 8; 8c‐cell, n = 8; 16‐cell, n = 145 and 12 for outer and inner cells, respectively; 32‐cell, n = 174 and 83 for outer and inner cells, respectively).

Figure 2

Effects of PAR‐aPKC system disruption on cell polarity and Yap localization. (A, B) Distribution of Yap and Pard6b proteins in embryos between the 8‐ and 32‐cell stages. (A) Distribution of Yap and Pard6b proteins in shEGFP‐injected embryos. (B) Distribution of Yap and Pard6b proteins in shPard6b‐injected embryos. Hoechst33258 (blue) was used to visualize nuclei. Dotted lines delineate the inner cells. (C) Distribution of Pard6b, PKC λ/ζ, and Scribble proteins in shEGFP and shPard6b‐injected embryos at the 16‐cell stage. (D) Distribution of F‐actin and p‐ERM in control (no injection) and shPard6b‐injected embryos at the 16‐ and 32‐cell stages. The number in each panel indicates the number of cells in the embryos. (E) Distribution of cortical F‐actin and p‐ERM in normal 16‐cell stage embryos. Stacked confocal images are shown. Note that the signals overlap in a dot‐like pattern. (F) Changes in the N/C ratio of Yap in the outer cells of shEGFP‐ and shPard6b‐injected embryos between the 8‐ and 32‐cell stages. Graphs show the mean ± SEM (n = 3 at each point except for shPard6b 16‐cell, where n = 4). ns, not significant; ****, P < 0.0001 vs. shEGFP‐injected embryos (two‐way ANOVA and Bonferroni's multiple comparisons test).

Figure 3

Effects of Par‐aPKC system disruption on Cdx2 expression. (A) Distribution of Cdx2 and Yap proteins in shEGFP‐ and shPard6b‐injected embryos. Nuclei were visualized with Hoechst33258. E‐cadherin was used to visualize the cell boundary. (B) Distribution of Cdx2 and Yap proteins in β‐globin‐ and dnPKC λ‐injected embryos. (C, D) Semi‐quantitative analyses of the effects of Par‐aPKC system disruption on the expression of Cdx2 at the 16‐cell stage. (C) Representative images of embryos showing strong, medium, and weak Cdx2 expression. Confocal images were projected to show total Cdx2 expression. (D) Graphs summarizing the expression of Cdx2 at the 16‐cell stage. The numbers in the graphs indicate the number of embryos in each category. (E) Distribution of PKC λ/ζ and Pard6b proteins in control and dnPKC λ‐injected embryos at the 16‐cell stage. (F, G) Semi‐quantitative analyses of the effects of Par‐aPKC system disruption on the expression of Cdx2 at the 32‐cell stage. (F) Representative images of embryos showing strong and weak Cdx2 expression. Confocal images were projected to show total Cdx2 expression. (G) Graphs summarizing the expression of Cdx2 at the 32‐cell stage. The numbers in the graphs indicate the number of embryos in each category.

Figure 4

Effects of Par‐aPKC system disruption on Hippo signaling at the 16‐cell stage. (A) Distribution of phosphorylated Yap (p‐Yap) and Yap proteins in 16‐cell stage embryos. The nuclei were stained with Hoechst33258. Dotted lines delineate the inner cells. (B, C) Intensity levels of p‐Yap signals (B) and N/C ratio of Yap (C) in the outer cells of the β‐globin‐ and dnPKC λ‐injected embryos shown in (A). (D, E) Intensity levels of p‐Yap signals (D) and N/C ratio of Yap (E) in the outer cells of the shEGFP‐ and shPar1a/b‐injected embryos shown in (A). (F) Semi‐quantitative analysis of the p‐Yap signals from β‐globin‐ and dnPKC λ‐injected embryos (n = 5 each). Graphs show the mean ± SEM. ns, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 vs. the control (β‐globin or shEGFP‐injected) embryos (t‐test).

Figure 5

16‐cell stage embryos often contain apolar cells in the outer position. (A–C) Distribution of Yap, p‐ERM, p‐Yap, and Amot in normal embryos at the 16‐cell stage. Arrowheads indicate apolar cells at the outer position. Dotted lines delineate the inner cells. (D) Distribution of the Yap N/C ratio of each blastomere in a representative 16‐cell stage embryo containing apolar outer cells. (E) Number of embryos with (+) or without (−) apolar outer cells.

Figure 6

Relationship between cell division, cell polarity, Hippo signaling, and cell position. (A) Representative images of 2/16 couplets generated by cell division of 1/8 blastomeres after a 10‐h incubation. Yap and p‐ERM are shown in green and red, respectively. Filled and open arrowheads indicate the nuclei of polar and apolar cells, respectively. (B) Representative images of 2/16 couplets generated by cell division of 1/8 blastomeres after a 14‐h incubation. The polar cell of the polar‐apolar couplets from a control embryo engulfed the apolar cell, in which Yap was clearly excluded from the nucleus (left panel). The bottom panel shows a confocal slice, 6 μm above the z‐plane of the couplet shown in the top panel. No engulfment was observed in the 2/16 couplets from dnPKC λ‐injected embryos (right panel). Yap and p‐ERM are shown in green and red, respectively. Filled and open arrowheads indicate the nuclei of polar and apolar cells, respectively. (C) The Yap N/C ratio in polar (n = 72) and apolar (n = 14) cells in the couplets after a 10‐h incubation. (D) Presence of inner cells in control (no injection, β‐globin‐, shEGFP ‐injected) and dnPKC λ‐, shPard6b‐, and shPar1a/b‐injected embryos. Solid and dotted lines delineate shapes of embryos and the inner cells, respectively. (E, F) Number of inner cells in embryos with disrupted Par‐aPKC systems at the 16‐cell stage. Inhibition of apical components by injection with dnPKC λ (n = 24) (E) and shPard6b (n = 26) (F) reduced the number of inner cells compared to that in β‐globin (n = 31) and shEGFP (n = 35) injected embryos, whereas inhibition of basolateral component by injection with shPar1a/b (n = 29) did not reduce the number of inner cells. Graphs show the mean ± SEM. ns, not significant; *, P < 0.05; ****, P < 0.0001 (C, t‐test; E and F, one‐way ANOVA and Tukey's multiple comparison test).

Figure 7

A model showing the relationship between the Par‐aPKC system, the cortical apical domain, represented by microvilli, and nuclear Yap. At the 16‐cell stage, both the Par‐aPKC system and cortical apical domain, represented by microvilli (p‐ERM/F‐actin), controls cell polarity and Yap localization. At the 32‐cell stage, the Par‐aPKC system plays a dominant role. See the Discussions for details.