The Apical Domain Is Required and Sufficient for the First Lineage Segregation in the Mouse Embryo

Abstract

Mammalian development begins with segregation of the extra-embryonic trophectoderm from the embryonic lineage in the blastocyst. While cell polarity and adhesion play key roles, the decisive cue driving this lineage segregation remains elusive. Here, to study symmetry breaking, we use a reduced system in which isolated blastomeres recapitulate the first lineage segregation. We find that in the 8-cell stage embryo, the apical domain recruits a spindle pole to ensure its differential distribution upon division. Daughter cells that inherit the apical domain adopt trophectoderm fate. However, the fate of apolar daughter cells depends on whether their position within the embryo facilitates apical domain formation by Cdh1-independent cell contact. Finally, we develop methods for transplanting apical domains and show that acquisition of this domain is not only required but also sufficient for the first lineage segregation. Thus, we provide mechanistic understanding that reconciles previous models for symmetry breaking in mouse development.

Keywords: apico-basal polarity; cell-fate specification; early mammalian development; reduced systems; self-organization; symmetry breaking.

Graphical abstract

Figure 1

Acquisition of the Apical Domain Predicts the First Lineage Segregation (A) Experimental design. Blastomeres (1/8-cell) are isolated from the 8-cell stage embryo into which mRNAs encoding fluorescent reporters are microinjected at the 2-cell stage. Development of “mini-blastocyst” recapitulates the TE versus ICM lineage segregation. (B) Time-lapse images of the developing 1/8-cell derived from Cdx2-EGFP × R26-H2B-mCherry embryo microinjected with Myr-palm-IFP (Memb) mRNA. The Cdx2 and Memb signals are adjusted differently in the last frame (20:15). (C) Time lapse of 1/8-cell isolated from R26-EGFP-Tuba embryo microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs. Dashed line denotes spindle. (D) Predominantly asymmetric 1/8-to-2/16-cell divisions as observed by spindle orientation relative to the apical domain (top; green line, random distribution; Kolmogorov-Smirnov test), and as defined by differential distribution of the apical domain (bottom). (E) Upon asymmetric division, polar cells envelop their apolar sister cells (top), the degree of which correlates with the relative level of Cdx2 expression (bottom; Spearman correlation). Arrowheads indicate the apical domain. Time, post-nuclear envelope breakdown (NEBD; hr:min). Scale bars, 10 μm. See also Figures S1 and S2; Movies S1 and S2.

Figure 2

Apical Domain Is Required for Lineage Segregation (A) A single-section immunofluorescence image of WT and mzPrkci^−/−;Prkcz^−/− E3.0 embryos simultaneously stained for Pard6b and Radixin. Intensity profile of Pard6b and Radixin is shown along the dashed lines. (B) A single-section immunofluorescence image of WT and mzCdc42^−/− E3.0 embryos simultaneously stained for aPKC, Pard6b, and Radixin. Intensity profile of aPKC, Pard6b, and Radixin is shown along the dashed lines. (C) A single-section immunofluorescence image of WT and mzPrkci^−/−;Prkcz^−/− E4.0 embryos simultaneously stained for Cdx2, Sox2, and DNA (DAPI). Scatter and density plots show fluorescence intensity of Cdx2 and Sox2 for individual blastomeres in WT (n = 605 cells pooled from 11 embryos) and mzPrkci^−/−;Prkcz^−/− (n = 350 cells pooled from 8 embryos) embryos; for Sox2 intensity p < 10⁻³⁹, Mann-Whitney U test. (D) A single-section immunofluorescence image of WT and mzCdc42^−/− E4.0 embryos simultaneously stained for Cdx2, Sox2, and DNA (DAPI). Scatter and density plots show fluorescence intensity of Cdx2 and Sox2 for individual blastomeres in WT (n = 605 cells pooled from 11 embryos) and mzCdc42^−/− (n = 149 cells pooled from 7 embryos) embryos; for Sox2 intensity p < 10⁻¹⁶, Mann-Whitney U test. Scale bars, 20 μm.

Figure 3

Apical Domain Controls Spindle Orientation (A) Time-lapse images of an asymmetric 8-to-16-cell division (white asterisk) of R26-EGFP-Tuba × mG embryo microinjected with Ezrin-mCherry mRNA, generating a polar (magenta asterisk) and apolar cell (green asterisk). Dashed line denotes spindle. Time, post-NEBD (hr:min). Right: quantification of apical domain distribution during 8-to-16-cell divisions. (B) Scatter plot showing the apical domain surface area and the angle between the spindle and the radial axis of the embryo for blastomeres undergoing 8-to-16-cell divisions in R26-EGFP-Tuba embryos microinjected with Ezrin-mCherry mRNA. Spearman correlation. (C) Spindle orientation of the 8-to-16-cell divisions relative to the radial axis of the embryo in WT, mzPrkci^−/−;Prkcz^−/−, and mzCdc42^−/− embryos (n = 130, 48 and 53 cells pooled from 17, 6, and 7 embryos, respectively). Green line, random distribution. Kolmogorov-Smirnov test. (D) Maximal-intensity projection (MIP) time-lapse images of the 8-cell stage SAS4-EGFP transgenic embryo microinjected with Ezrin-mCherry mRNA. Time: 00:00 is 68 hr post-hCG (hr:min). (E) MIP live images of the 16-cell stage SAS4-EGFP × mT, mzPrkci^−/−;Prkcz^−/− × SAS4-EGFP × mT and mzCdc42^−/− × SAS4-EGFP × mT embryos. Arrow points to off-centered MTOC cluster. Arrowheads indicate the apical domain. Scale bars, 20 μm. See also Figures S3 and S4; Movies S3 and S4.

Figure 4

Apical Domain Is Sufficient for Initiating Cell-Fate Segregation (A) Experimental design. The apical domain is transplanted into an apolar 8-cell stage blastomere to test whether it can induce the cell lineage segregation process. (B) Time lapse of an 8-cell stage blastomere isolated from Cdx2-EGFP embryo microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs, developing after integration of a cell fragment (white asterisk) derived from an 8-cell stage blastomere containing the apical domain (note Cdx2 expression marked by yellow asterisk). Arrowheads indicate the apical domain. (C) Time lapse of an 8-cell stage blastomere isolated from Cdx2-EGFP embryo microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs, developing after integration of a cell fragment (asterisk) derived from an 8-cell stage blastomere containing the non-apical domain. Time, post-NEBD (hr:min). Scale bars, 10 μm. See also Movie S5.

Figure 5

Apical Domain Controls Cell Fate through Yap Signaling (A) Schematic and immunofluorescence staining for subcellular distribution of Amot, Yap, and Radixin (apical domain) in the 16-cell stage embryo and in a 2/16-doublet, respectively. (B) Experimental design. The apical domain is transplanted into an apolar cell of 2/16-doublet to test whether it can change subcellular distribution of Amot and Yap. (C) Time lapse of a 2/16-cell doublet isolated from the embryo microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs, developing after integration of a cell fragment (white asterisk) derived from an 8-cell stage blastomere containing the apical domain. (D) Immunofluorescence image of the 2/16-cell doublet after transplantation of apical domain (recipient cell marked by asterisk) stained for Amot, Yap, Radixin, and DNA (DAPI). (E) Time lapse of a 2/16-cell doublet isolated from the embryo microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs, developing after integration of a cell fragment (asterisk) derived from an 8-cell stage blastomere containing the non-apical domain. (F) Immunofluorescence image of the 2/16-cell doublet after transplantation of the non-apical domain (recipient cell marked by asterisk) stained for Amot, Yap, Radixin, and DNA (DAPI). (G) Cortical intensity profiles under the dashed line of apolar recipient cell shown in (A), (D), and (F). (H) Box plot showing the nucleus-to-cytoplasm (n/c) Yap intensity ratio of polar cell divided by that of its sister cell in the respective 2/16-cell doublet. Mann-Whitney U test. In the box plot, the central mark indicates the median, with the bottom and top edges of the box indicating the 25^th and 75^th percentiles, respectively. The whiskers extend to the most extreme data points, with exception of the outliers that are marked individually with the + symbol. n.s., not significant. Arrowheads indicate the apical domain. Time, post-fusion (min:s). Scale bars, 10 μm.

Figure 6

Cell Position Regulates Apical Domain Formation and Fate Specification (A) Time lapse of asymmetric 8-to-16-cell division (white asterisk; generating cells marked with orange and magenta asterisks) of Cdx2-EGFP × R26-H2B-mCherry embryo microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs, in which an apolar cell (orange asterisk) acquires the apical domain (from yellow to white arrowheads) and begins expressing Cdx2. Dynamics of Cdx2 expression in (top) an 8-cell stage blastomere undergoing asymmetric division generating one TE- and the other ICM-forming cell, and (bottom) another 8-cell stage example, as shown in the top panels, with line colors indicating the cells marked by asterisks of the same color. Cdx2 expression is upregulated (black arrowhead) after an apolar cell acquires the apical domain (red underline). (B) Time lapse of the halved embryo in which an apolar cell generated after 4/8-to-8/16-cell division (white asterisk; generating cells marked with orange and magenta asterisks) acquires the apical domain (from yellow to white arrowheads). The Memb signals are adjusted differently in the last frame (09:50). The ratio of initially apolar cells that acquire the apical domain is different between the whole, halved, and 2/16-embryos. Two-tailed Fisher's exact test. Magenta asterisk, polar cell. Time, post-NEBD (hr:min). Scale bars, 20 μm. See also Figure S5.

Figure 7

Cdh1-Independent Cell Contact Directs Apical Domain Formation (A) Cdh1-coated beads induce the apical domain opposite to the contact in WT 1/8-cell, as well as mzCdh1^−/− cell, microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs. PMMA beads also induce the apical domain in mzCdh1^−/− cells. Time, post-NEBD (hr:min). Scale bars, 10 μm. Lower panels: quantification of apical domain position relative to the contact with a bead. Green line, random distribution. Kolmogorov-Smirnov test. (B) Live image of the 8-cell stage mzCdh1^−/− embryo microinjected with Ezrin-mCherry and Myr-palm-IFP mRNAs. Scale bar, 20 μm. (C) Model of symmetry breaking in mouse development. The presence of contact-free cell surface in outside cells directs formation of the apical domain that, in turn, induces asymmetric division and TE-fate specification. Arrowheads indicate the apical domain. See also Figure S6 and Movie S6.