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