Rho-associated protein kinase regulates subcellular localisation of Angiomotin and Hippo-signalling during preimplantation mouse embryo development  Aleksandar I. Mihajlović, Alexander W. Bruce  Reproductive BioMedicine Online  Volume 33, Issue 3, Pages 381-390 (September 2016) DOI: 10.1016/j.rbmo.2016.06.028 Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 1 Preimplantation mouse embryo development after Rock-inhibition. A) Schematic representation of experimental strategy to assay in-vitro development of preimplantation mouse embryos in three different concentrations of Rock-inhibitor Y-27632 (+dimethylsulphoxide (DMSO) controls). B) Representative bright-field micrographs of inhibitor- and vehicle-treated embryos at the end of the culture period. Lower panels depict embryos cultured in increasing concentrations of Rock-inhibitor, with appropriate DMSO vehicle control embryos displayed in corresponding upper panels. Note the titration effect of Rock-inhibition and pronounced cell death in 50 µmol/l and 100 µmol/l treated embryos. Scale bar = 100 µm. Reproductive BioMedicine Online 2016 33, 381-390DOI: (10.1016/j.rbmo.2016.06.028) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 2 Rock-inhibition is associated with defective apical-basolateral polarity and tight junction formation. A) Schematic representation of experimental strategy to assay subcellular expression of stated cytoskeletal, apical-basolateral polarity-associated or junctional proteins in control or Y-27632-treated (50 µmol/l)/ Rock-inhibited embryos at 32-cell stage. B) Representative single z-section confocal micrographs, plus selected projected images, of embryos immunofluorescently stained for stated proteins (grey-scale) and DNA (DAPI counter-stain, magenta). Note, ‘AJs’ and ‘TJs’ relate to images immuno-stained for ‘adherens’ and ‘tight’ junction markers, respectively. ‘PB’ denotes the second meiotic polar body. In Rock-inhibited embryos, yellow arrows highlight atypical marker protein localisation, versus controls. Yellow arrowhead in pERM immunostained projection denotes atypically enriched ‘apical-disc’ formation in Rock-inhibited embryos. Blue arrows in the control group denote tight-junction-associated actin and Tjp2 and blue asterisks mark lack of tight-junction-localised actin in Rock-inhibited embryos. Scale bar = 20 µm. Reproductive BioMedicine Online 2016 33, 381-390DOI: (10.1016/j.rbmo.2016.06.028) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 3 Enhanced and mislocalised outer cell Amot expression in Rock-inhibited embryos, is related to activated Hippo-signalling. A) Schematic representation of experimental strategy to assay Amot protein expression and localisation in combination with either Yap1 or Ctnnb1, in 32-cell stage control or Y-27632-treated (50 µmol/l)/ Rock-inhibited embryos. B) Representative single z-section confocal micrographs of control and Rock-inhibited embryos, double-immunofluorescently stained for Amot (grey-scale) and either Yap1 or Ctnnb1 (green), with DAPI DNA counter-stain (magenta). Yellow, white and blue arrows highlight atypical protein localisation in Rock-inhibited embryos, versus controls, for Amot (outer cell lateral domains), Yap1 (outer-cell-cytoplasmic localisation) and Ctnnb1 (outer cell apical-domain enrichment), respectively. n.b., enhanced overall Amot expression in outer cells of Rock-inhibited embryos versus controls. Scale bar = 20 µm. C) Graphical representation of averaged percentages of outer and inner cells exhibiting nuclear Yap1 signal less than cytoplasmic signal (N < C), nuclear Yap1 signal greater than cytoplasmic signal (N > C) or approximately equal signals (N = C), from control (n = 14) or Rock-inhibited (n = 12) embryos. Reproductive BioMedicine Online 2016 33, 381-390DOI: (10.1016/j.rbmo.2016.06.028) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions

Figure 4 Rock-inhibition induced ectopic outer cell Hippo-signalling activation, is mediated by Amot. A) Experimental strategy to knockdown Amot protein expression in developing preimplantation control (dimethylsulphoxide [DMSO]) or Rock-inhibited (Y-27632, 50 µmol/l) embryos, using control (GFP-dsRNA) or Amot-specific (Amot-dsRNA) RNAi. B) Representative single z-section confocal micrographs of GFP-dsRNA or Amot-dsRNA microinjected, DMSO control or Rock-inhibitor (Y-27632) treated embryos, double-immunofluorescently stained for Amot (grey-scale) and Yap1 (green), upper panels, or Pard6b (grey-scale) and Yap1 (green), lower panels. DNA DAPI counter-stain and RDBs (microinjection marker) signal, pseudo-coloured magenta and yellow, respectively. Relating to Rock-inhibited GFP-dsRNA microinjected embryos, yellow and blue arrows denote atypical basolateral localisation of Amot and Pard6b, versus DMSO vehicle treated embryos, (similar to Figures 2 and 3); white arrows highlight outer cells no longer exhibiting nuclear enriched Yap1 (similar to Figure 3); thus confirming the dependency of appropriate apical polarization and Hippo-signalling suppression on Rock activity in outer cells. Regarding Amot-dsRNA microinjected embryos, white asterisks and arrowheads highlight nuclear enriched Yap1 that is present in both inner and outer cells, respectively, irrespective of Rock-inhibition status (contrast with GFP-dsRNA microinjected embryos). Moreover, blue arrows again mark atypical Pard6b basolateral expression in outer cells, only after Rock-inhibition. Therefore, confirming Rock-activity mediated effects on Hippo-signalling act through the regulation of apical-basolateral polarization (assayed by Pard6b localisation) and then via the Amot protein (as depletion of Amot is able to block Rock-inhibitor mediated activation of the Hippo-signalling pathway, in outer cells, assayed by Yap1 localisation, despite apical-basolateral polarity defects). ‘PB’ denotes second meiotic polar body. Scale bar = 20 µm. Reproductive BioMedicine Online 2016 33, 381-390DOI: (10.1016/j.rbmo.2016.06.028) Copyright © 2016 Reproductive Healthcare Ltd. Terms and Conditions