Actomyosin polarisation through PLC-PKC triggers symmetry breaking of the mouse embryo

Abstract

Establishment of cell polarity in the mammalian embryo is fundamental for the first cell fate decision that sets aside progenitor cells for both the new organism and the placenta. Yet the sequence of events and molecular mechanism that trigger this process remain unknown. Here, we show that de novo polarisation of the mouse embryo occurs in two distinct phases at the 8-cell stage. In the first phase, an apical actomyosin network is formed. This is a pre-requisite for the second phase, in which the Par complex localises to the apical domain, excluding actomyosin and forming a mature apical cap. Using a variety of approaches, we also show that phospholipase C-mediated PIP₂ hydrolysis is necessary and sufficient to trigger the polarisation of actomyosin through the Rho-mediated recruitment of myosin II to the apical cortex. Together, these results reveal the molecular framework that triggers de novo polarisation of the mouse embryo.The molecular trigger that establishes cell polarity in the mammalian embryo is unclear. Here, the authors show that de novo polarisation of the mouse embryo at the 8-cell stage is directed by Phospholipase C and Protein kinase C and occurs in two phases: polarisation of actomyosin followed by the Par complex.

Fig. 1

Dynamics of actomyosin and Pard6 polarisation at the 8-cell stage. a Mouse embryos were fixed at early, mid and late 8-cell stage, and immunostained for F-actin and Pard6. Arrowheads indicate the magnified blastomeres. b Circumferential line profiles of F-actin and Pard6 fluorescence intensity in embryos from (a). Yellow indicates cell–cell contact regions and blue cell-contact-free domain. Smoothened data were displayed as coloured curves to show the pattern of the signal. c Cell-contact-free enrichment of F-actin and Pard6 plotted against the IEA in embryos from (a). Each dot represents an individual measurement. N = 25 embryos, three independent experiments. LOWESS plot were used to display the tendency of different datasets. The shadow indicates the 95% confidence interval. d Summary of the different phases of polarisation during the 8-cell stage. During the first phase, embryos start compacting and actomyosin becomes polarised to the apical domain. During the second phase, embryos are already compacted and apical domain components, including Pard6 polarise apically. All scale bars, 15 μm

Fig. 2

A polarised and active actomyosin network is required for Par complex polarisation. a 8-cell stage (blebbistatin, LatB group and its control) or 4–8 cell stage embryos (ML-7 and its control) were treated with DMSO, blebbistatin, ML-7 or LatB, and fixed at the late 8-cell stage. Early 8-cell stage embryos treated with DMSO were also fixed as a control. Embryos were immunostained for F-actin and Pard6. b, c Cell-contact-free enrichment of F-actin (b) or Pard6 (c) as a function of the IEA in embryos from (a). Each dot represents an individual measurement. d Percentage of polarised embryos in the groups of (a). The number of embryos per category is indicated. Embryos that did not show any blastomere with the enrichment of F-actin and Pard6 at the cell-contact-free surface were considered non-polarised. Otherwise embryos were classified as polarised. Data are shown as a contingency table. ****p < 0.0001, NS = not significantly different, Fisher’s exact test (six independent experiments). e DMSO and ML-7-treated embryos immunostained for ppMRLC. f Quantification of ppMRLC cell-contact-free cortical enrichment in ML-7-treated and control embryos. Each dot represents an individual measurement. Data are shown as individual data points with Box and Whiskers graph (bottom: 25%; top: 75%; line: median; whiskers: min to max). ****p < 0.0001, Mann–Whitney test. N = 10 embryos for DMSO and N = 12 embryos for ML-7, four independent experiments. g ppMRLC and F-actin staining from the late 4-cell stage to the late 8-cell stage. The ppMRLC signal intensity of the indicated blastomeres is plotted in 3D. White stars indicate the cell-contact-free surface. Arrowheads indicate cortical localisation of ppMRLC. h Quantification of ppMRLC enrichment at the cell-contact and cell-contact-free domains from the late 4-cell stage to the late 8-cell stage. Data are shown as mean ± s.e.m. For all panels, squares indicate the magnified regions. N = 32 measurements from 7 late 4-cell stage embryos; N = 69 measurements from 11 early 8-cell stage embryos; N = 86 measurements from 11 mid 8-cell stage embryos; N = 50 measurements from 13 late 8-cell stage. All scale bars, 15 μm

Fig. 3

PLC–PKC activity is essential for polarisation at the 8-cell stage. a Scheme of the inhibitor (sphingosine, calphostin C) treatment. (b) DMSO, sphingosine and calphostin C-treated embryos immunostained for F-actin and Pard6. c, d Cell-contact-free enrichment of F-actin (c) or Pard6 (d) as a function of the IEA in embryos from (b). Each dot represents an individual measurement. e Percentage of polarised embryos in the groups of (b). The number of embryos per category is indicated. Embryos that did not show any blastomere with the enrichment of F-actin and Pard6 to the cell-contact-free surface were considered non-polarised. Otherwise embryos were classified as polarised. Data are shown as a contingency table. ****p < 0.0001, Fisher’s exact test. f Quantification of polarisation in embryos from (b). Data are shown as mean ± s.d. ****p < 0.0001, unpaired two-tailed Student’s t test (three independent experiments). g Scheme of U73122 treatment and OAG rescue experiment. h DMSO, U73122 and U73122 + OAG-treated embryos immunostained for F-actin and Pard6. i, j Cell-contact-free enrichment of F-actin (i) or Pard6 (j) as a function of the IEA in embryos from panel h. Each dot represents an individual measurement. k Percentage of polarised embryos in the groups of (h). The number of embryos per category is indicated. Data are shown as a contingency table. ****p < 0.0001, NS = not significantly different, Fisher’s exact test. l Quantification of polarisation in embryos from (h). Data are shown as mean ± s.d. ***p < 0.001, ****p < 0.0001, NS = not significant different, Kruskal–Wallis test and Dunn’s multiple comparisons test (two independent experiments). m Scheme of the PLC-DN overexpression and OAG rescue experiment. n Ruby and PLC-DN overexpressing embryos ± OAG immunostained for F-actin and Pard6. o Percentage of polarised embryos in the groups of (n). The number of embryos per category is indicated. Data are shown as a contingency table. ****p < 0.0001, **p < 0.01, Fisher’s exact test. p Quantification of polarisation in embryos from (n). Data are shown as mean ± s.d. **** p < 0.0001, **p < 0.01, *p < 0.05, Kruskal–Wallis and Dunn’s multiple comparisons tests (two independent experiments). All scale bars, 15 μm

Fig. 4

PLC–PKC inhibition abolishes apical ppMRLC accumulation. a DMSO and sphingosine-treated embryos stained for F-actin and ppMRLC (b). Quantification of ppMRLC enrichment at the cell-contact-free domain in embryos from (a) N = 9–10 embryos, two independent experiments. c DMSO and calphostin C-treated embryos stained for F-actin and ppMRLC. d Quantification of ppMRLC enrichment at the cell-contact-free domain in embryos from (c). N = 11 embryos for DMSO and N = 9 embryos for calphostin C, two independent experiments. e DMSO and U73122-treated embryos stained for F-actin and ppMRLC. f Quantification of ppMRLC cell-contact-free cortical enrichment in embryos from (e). N = 20 embryos for DMSO and N = 22 embryos for U73122, three independent experiments. g Ruby and PLC-DN-overexpressing embryos stained for F-actin and ppMRLC. h Quantification of ppMRLC cell-contact-free cortical enrichment enrichment in embryos from (g). N = 7 embryos for Ruby and N = 8 embryos for PLC-DN, two independent experiments. ****p < 0.0001, Mann–Whitney test. For all panels, arrowheads indicate apically localised ppMRLC and squares indicate the magnified regions. Data are shown as individual data points with Box and Whiskers graph (bottom: 25%; top: 75%; line: median; whiskers: min to max). All scale bars, 15 μm

Fig. 5

Ectopic activation of PKC expands the apical domain and induces premature actomyosin polarisation. a 8-cell stage embryos treated with DMSO or OAG and immunostained for F-actin and Pard6. Yellow arrows indicate the borders of the cell-contact-free domain and white arrows indicate the borders of the Pard6 apical domain. b Quantification of the Pard6 surface coverage rate in embryos from (a). The surface coverage rate is calculated as the ratio between the length of Pard6 positive domain and the length of the cell-contact-free surface. Data are shown as individual data points with Box and Whiskers graph. ****p < 0.0001, two-tailed unpaired Student’s t test. N = 8 embryos for DMSO and N = 6 embryos for OAG, four independent experiments. c 4-cell stage embryos treated with DMSO or OAG and immunostained for F-actin, ppMRLC and Pard6. Arrowheads indicate apically polarised F-actin or ppMRLC. d Cell-contact-free enrichment of F-actin and Pard6 as a function of the IEA in embryos from (c). Dots represent individual measurements. e Quantification of cell-contact-free cortical enrichment of ppMRLC in embryos from (c). Data are shown as individual data points with a Box and Whiskers graph (bottom: 25%; top: 75%; line: median; whiskers: min to max). ****p < 0.0001, Mann–Whitney test. N = 18 embryos for DMSO and N = 10 embryos for OAG, six independent experiments. f 4-cell stage embryos expressing GFP-MRLC or GFP-MRLC + PKCɑ-A25E in two blastomeres were immunostained for GFP, F-actin and Pard6. Arrowheads indicate the injected blastomeres. g, h Quantification of cell-contact-free enrichment of GFP-MRLC (g) or F-actin (h) in embryos from (f). Data are shown as individual data points with Box and Whiskers graph (bottom: 25%; top: 75%; line: median; whiskers: min to max). ***p < 0.001, ****p < 0.0001, Mann–Whitney test. N = 6 embryos, three independent experiments. i Scheme of the PKC-localised activation using the CRY2-CIB1 photoactivatable system. j Time-lapse snapshots of the localisation of CRY2-PKC-KD under localised blue-light illumination. k Blastomeres expressing CIB1-Zsgreen-CAAX and CRY2-PKC-KD were regionally illuminated using a 458 nm wavelength and immunostained for F-actin, ppMRLC and Pard6. Arrowheads or dotted circles indicate the illuminated region. Squares indicate the magnified regions. (N = 24 embryos, 11 independent experiments). All scale bars, 15 μm

Fig. 6

PLC–PKC inhibition prevents myosin II apical recruitment independently of myosin phosphorylation. a GFP-MRLC or GFP-MRLC-DD overexpressing embryos were injected with Ruby or PLC-DN and fixed at the late 8-cell stage. b Percentage of blastomeres showing a polarised GFP-MRLC or GFP-MRLC-DD in the groups of (a). N = 17 embryos for Ruby + MRLC, N = 17 embryos for PLC-DN + MRLC and N = 17 embryos for PLC-DN + MRLC-DD, three independent experiments. c Scheme of PLC–PKC inhibitor treatment experiments. d GFP-MRLC or GFP-MRLC-DD overexpressing embryos were treated with DMSO or U73122 and fixed at the late 8-cell stage. e Percentage of blastomeres showing a polarised GFP-MRLC or GFP-MRLC-DD in the groups of (d). N = 11 embryos for DMSO + MRLC, N = 11 embryos for U73122 + MRLC and N = 9 embryos for U73122 + MRLC-DD, four independent experiments. f GFP-MRLC or GFP-MRLC-DD overexpressing embryos were treated with DMSO or sphingosine and fixed at the late 8-cell stage. g Percentage of blastomeres showing a polarised GFP-MRLC or GFP-MRLC-DD in the groups of (f). N = 20 embryos for DMSO + MRLC, N = 9 embryos for sphingosine + MRLC and N = 20 embryos for sphingosine + MRLC-DD, two independent experiments. h GFP-MRLC or GFP-MRLC-DD overexpressing embryos were treated with DMSO or ML-7 and fixed at the late 8-cell stage. i Percentage of polarised blastomeres in the groups of (h). N = 18 embryos for DMSO + MRLC, N = 17 embryos for ML-7 + MRLC, N = 15 embryos for DMSO + MRLC-DD, N = 18 embryos for ML-7 + MRLC-DD, two independent experiments. For all, the quantifications data are shown as a contingency table and the n number in each bar indicates the total number of blastomeres analysed. Squares indicate the magnified regions. ****p < 0.0001, NS = not significantly different, Fisher’s exact test. All scale bars, 15 µm

Fig. 7

RhoA mediates MRLC and Pard6 cortical polarisation downstream of PLC–PKC signalling. a Scheme of the PLC–PKC rescue experiment using constitutively active Rho (RhoA-Q63L). b GFP-MRLC overexpressing embryos were injected with Ruby or PLC-DN and immunostained for GFP and Pard6 at the 8-cell stage. Arrowheads indicate the apical domain. c, d Percentage of polarised blastomeres based on either Pard6 (c) or GFP-MRLC (d) localisation in the groups of (b). N = 17 embryos for Ruby, N = 17 embryos for PLC-DN, N = 18 embryos for PLC-DN + RhoA-Q63L, two independent experiments. e Scheme of the PLC–PKC rescue experiment using constitutively active Rho (RhoA-Q63L). f GFP-MRLC overexpressing embryos were treated with DMSO or U73122 and immunostained for GFP and Pard6 at the 8-cell stage. Arrowheads indicate the apical domain. g, h Percentage of polarised blastomeres based on either Pard6 (g) or GFP-MRLC (h) localisation in the groups of (f). N = 21 embryos for DMSO, N = 17 embryos for U73122, N = 27 embryos for U73122 + RhoA-Q63L. i Summary model of the signalling events that trigger symmetry breaking and polarisation at the 8-cell stage. PLC-mediated PIP₂ hydrolysis activates PKC to trigger the apical enrichment of myosin II, a subsequent reorganisation of the cortical cytoskeleton and symmetry breaking. The apical polarisation of myosin II requires both RhoA activation (downstream of PLC–PKC signalling) and MLCK-mediated MRLC di-phosphorylation. At the late 8-cell stage, the actin-myosin enriched apical cortex recruits Pard6 (directly or indirectly) to the apical domain. For all the quantifications, data are shown as a contingency table and the n number in each bar indicates the total number of blastomeres analysed. ****p < 0.0001, NS = not significantly different, Fisher’s exact test. Squares indicate the magnified regions. All scale bars, 15 µm