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During mouse pre-implantation development, extra-embryonic primitive endoderm (PrE) and pluripotent epiblast precursors are specified in the inner cell mass (ICM) of the early blastocyst in a 'salt and pepper' manner, and are subsequently sorted into two distinct layers. Positional cues provided by the blastocyst cavity are thought to be instrumental for cell sorting; however, the sequence of events and the mechanisms that control this segregation remain unknown. Here, we show that atypical protein kinase C (aPKC), a protein associated with apicobasal polarity, is specifically enriched in PrE precursors in the ICM prior to cell sorting and prior to overt signs of cell polarisation. aPKC adopts a polarised localisation in PrE cells only after they reach the blastocyst cavity and form a mature epithelium, in a process that is dependent on FGF signalling. To assess the role of aPKC in PrE formation, we interfered with its activity using either chemical inhibition or RNAi knockdown. We show that inhibition of aPKC from the mid blastocyst stage not only prevents sorting of PrE precursors into a polarised monolayer but concomitantly affects the maturation of PrE precursors. Our results suggest that the processes of PrE and epiblast segregation, and cell fate progression are interdependent, and place aPKC as a central player in the segregation of epiblast and PrE progenitors in the mouse blastocyst.
Fig. 1. aPKC is enriched in PrE precursors from the mid blastocyst. (A) Immunofluorescence for aPKC at successive stages of pre-implantation development. (B-Dâ²) Quantification of aPKC levels from mid- to E4.5 blastocysts. Red lines/bars indicate PrE precursors (GATA4+); blue lines/bars indicate epiblast precursors (GATA4-). (Dâ²) Quantification of aPKC levels along apical (grey/white) and basal (black) membranes of the PrE at E4.5. Histograms compare fluorescence intensity between both ROIs on the ICM depicted. Bar charts compare averages from several measurements. Dashed lines on histograms represent averages for profiles shown. (E) Immunofluorescence for aPKC and E-cadherin on an early-mid blastocyst showing aPKC localisation on the membrane/cortex. (F) Immunofluorescence for aPKC and E-cadherin at E4.5. There is a low aPKC staining throughout the ICM, except on the apical membrane of PrE cells. (G) Colocalisation of aPKC and DAB2 at E4.5. Histogram shows overlay of aPKC and DAB2 levels on the pictures above. **P<0.01, ***P<0.001, unpaired Studentâs t-test. Bar charts display mean+s.e.m. n, number of embryos. Scale bars: 20 μm.
Fig. 2. aPKC enrichment and polarisation are downstream of FGF/ERK signalling. (A) Schematic of 2i or Fgf4 treatment. (B) Number of GATA4 cells in control and 2i conditions. (C) Number of polarised cells on the ICM surface per embryo. 20/20 control embryos had at least eight cells. 23/26 2i-treated embryos had 0-6 polarised cells. (D) Number of GATA4 cells in control and Fgf4-treated embryos. (E-H) Immunofluorescence for aPKC and GATA4 in the ICM. (E) Higher levels of aPKC in PrE cells of control embryos after 48 hours (arrows), but not in those treated with 2i (F). (G) Polarised apical aPKC in PrE cells in controls after 72 hours (arrowheads), but not in 2i-treated embryos (H). (I-M) Immunofluorescence as in E-H for Fgf4 treatment. Both control (I,K) and experimental (J,L,M) embryos have apical aPKC in cells on the ICM surface (arrowheads). All data are collected from three replicas of each experiment. **P<0.01, ***P<0.001, unpaired Studentâs t-test. Bar charts display mean+s.e.m. n, number of embryos. Scale bars: 20 μm.
Fig. 3. Knockdown of aPKC prevents PrE cells from reaching the ICM surface. (A) Live images 24 hours post-injection (hpi). Arrowheads indicate cells on the ICM surface in control embryo. Dashed line indicates inner cells in experimental embryo. (B) Percentage of labelled ICM cells (H2B-GFP+) per embryo at 24 hpi. (C) Percentage of labelled ICM cells per embryo at 48 hpi. (D) Percentage of labelled PrE cells (GATA4+) per embryo at 48 hpi. (E) Percentage of labelled PrE cells (GATA4+) on the ICM surface per sorted embryo at 48 hpi. (F-H) Immunofluorescence for aPKC and GATA4 in representative embryos. Arrowheads indicate labelled GATA4+ cells. Asterisks indicate labelled GATA4-cells inside the ICM. Insets show the detail of ICMs. Each channel also overlaid with nuclear staining in all panels. **P<0.01; ***P<0.001, unpaired Studentâs t-test (B,C) or Mann-Whitney U-test (D,E). Charts display mean+s.e.m. In scatter dot plot, each dot represents one embryo. n, number of embryos. Scale bars: 20 μm.
Fig. 4. Inhibition of aPKC in mid blastocysts impairs the segregation of PrE and epiblast. (A) Schematic of PKC inhibitor treatment. (B) Snapshots of a control embryo cultured in 1% DMSO. (C,D) Snapshots of two embryos cultured in 5 μM Gö6983. See supplementary material Movies 4-6, respectively. Green fluorescence indicates H2B-GFP expressed from the Pdgfra locus (PrE precursors). (E) Percentage of sorted embryos: 89% of embryos have a sorted PrE layer after 1% DMSO treatment, 37% of embryos have a sorted PrE layer after 5 μM Gö6983 treatment (P<0.0001, Fisherâs exact test; see Table 1). (F) Time course of PrE expansion. Each line represents one embryo (number of GFP+ cells over time). Bold lines correspond to embryos in B-D as indicated (asterisks). (G) Number of apoptoses per cell division. (H) PrE (GFP+) expansion from the beginning to the end of the culture. **P<0.01, unpaired Studentâs t-test. Bar charts display mean+s.e.m. Scale bars: 20 μm.
Fig. 5. Inhibition of aPKC prevents the maturation of PrE identity. (A-Bâ²) Late blastocysts, live (A,B) and stained for GATAA4 and OCT4 after culture in 1% DMSO (Aâ²) or 5 μM Gö6983 (Bâ²). GATA4 is present beyond the nuclear borders after treatment with Gö6983. See also supplementary material Movies 7-9. (C) Total, TE and ICM cell numbers. n=17 embryos for 1% DMSO, n=13 for 5 μM Gö6983. (D) Cell numbers for each ICM lineage. n=41 embryos for 1% DMSO, n=36 for 5 μM Gö6983. (E-H) Immunofluorescence for aPKC, DAB2 and GATA4 (E,F) or LRP2 and GATA4 (G,H). Insets show detail of ICMs. Each channel also overlaid with nuclear staining in all panels. ***P<0.001, unpaired Studentâs t-test. Bar charts display mean±s.e.m. Scale bars: 20 μm.
Fig. 6. Inhibition of aPKC prevents the maturation of PrE identity. (A,B) Immunofluorescence for GATA4 and NANOG in representative embryos after culture in 1% DMSO or 5 μM Gö6983. Asterisks indicate double-negative cells, with no staining for either NANOG or GATA4. (C,D) Immunofluorescence for GATA6 and NANOG as in A,B. Arrow indicates a NANOG+, GATA6+ cell on the ICM surface after Gö6983 treatment. (E) Immunofluorescence for GATA4 and OCT4 after treatment with 5 μM Gö6983. GATA4 is excluded from the nuclei present in the cytoplasm throughout the ICM. (F) Immunofluorescence for GATA4 and NANOG after treatment with 5 μM Gö6983. GATA4 is distributed throughout the cell marked with arrows (nucleus and cytoplasm). (G) Immunofluorescence for GATA4 and GATA6 after treatment with 10 μM Gö6983. Arrowheads indicate polar TE cells that retain GATA6 (normally found only in PrE and mural TE at this stage).
Fig. 7. Proposed model for PrE formation. ICM cells become primed towards a PrE or epiblast fate through the action of paracrine Fgf4. Sustained production of Fgf4 by epiblast precursors reinforces PrE fate, which is accompanied by aPKC enrichment in PrE precursors and upregulation of GATA4. aPKC would trigger survival signals in PrE cells that reach the ICM surface, which maintain their position. aPKC becomes polarised on the apical side of PrE cells at E4.5, when the PrE forms an epithelium.
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