Current Biology, Volume 23 Supplemental Information Polarity-Dependent Distribution of Angiomotin Localizes Hippo Signaling in Preimplantation Embryos Yoshikazu Hirate, Shino Hirahara, Ken-ichi Inoue, Atsushi Suzuki, Vernadeth B. Alarcon, Kazunori Akimoto, Takaaki Hirai, Takeshi Hara, Makoto Adachi, Kazuhiro Chida, Shigeo Ohno, Yusuke Marikawa, Kazuki Nakao, Akihiko Shimono, and Hiroshi Sasaki

Author Contributions Y. Hirate performed most of the experiments. Y. Hirate and H. Sasaki designed the research and wrote the paper. S. Hirahara, K.-I. Inoue, and K. Nakao performed the analysis of the Par–aPKC system in embryos. A. Shimono demonstrated the interaction between Amot and Yap and provided advice and reagents for the analysis of Amot. A. Suzuki provided advice and reagents for the analysis of cell polarity. V.B. Alarcon and Y. Marikawa developed the shPard6b knockdown system and provided protocols and reagents prior to publication. K. Akimoto and S. Ohno provided the PKC mutant mouse and shared unpublished information on the phenotypes of PKC double-mutant embryos. T. Hirai, T. Hara, and K. Chida generated the PKC mutant mouse and also provided Kibra cDNA. M. Adachi provided reagents and advice for the analysis of Amotl2.

Figure S1. Effects of Disrupting the Apical Par–aPKC System on Hippo Signaling in 32-Cell Stage Embryos, Related to Figure 1

(A) Distribution of apical and basolateral proteins in Pard6b KD embryos. Injection of shPard6b reduced apically localized Pard6b and p-ERM proteins. The basolateral proteins Scribble and Lgl1 redistributed to the outer membrane (dots).

(B) Distribution of apical and basolateral proteins in embryos overexpressing dnPKC mediated by RNA injection at the two-cell stage. Around the 32-cell stage, overexpressed PKC was distributed throughout the plasma membrane (n = 3/3). As a result, endogenous Pard6b was probably also distributed throughout the plasma membrane (n = 7/7). Although this result was slightly different from that observed with Pard6b KD embryos, the effects of dnPKC were essentially the same. The apical domain was disrupted, as demonstrated by clear reduction in the p-ERM signal (n = 4/4). Basolateral domains were expanded, as shown by the unrestricted distribution of Scribble (n = 7/7), Lgl1 (n = 9/9), and E-cadherin (n = 7/7, data not shown) proteins. dnPKCembryos also exhibited reduced nuclear Yap, increased p-Yap, and reduced Cdx2 expression in the outer cells.

(C) Strategy for the targeted disruption of PKC The wild-type allele of PKC including exons 6–10 (boxes), the targeting vector, the floxed allele, and the mutated allele produced by Cre recombinase are shown. The restriction endonuclease sites were as follows: Hi, HindIII; Ap, ApaI. The LoxP sites are indicated by the open triangles. The recognition sites for the PCR primers used in the genotyping experiments shown in panel E are indicated by arrowheads.

(D) Southern blot analysis of genomic DNA from wild-type and PKC-floxed ES cells. The HindIII-digested fragments of 11.5 kb and 7.0 kb were derived from wild-type and floxed alleles, respectively. The ApaI-digested fragments of 11.3 kb and 7.5 kb were derived from wild-type and targeted alleles, respectively.

(E) PCR genotyping of wild-type, PKC+/−, and PKC−/− mice. Amplicons of 1,982 bp and 322 bp were derived from the wild-type and mutant alleles, respectively.

(F) Analysis of embryos obtained by intercrossing PKC+/−:PKC+/− mice. Representative embryos obtained by intercrossing PKC+/−:PKC+/− mice. Anti-PKCantibody was used to detect PKC and PKCNote that the abnormalities observed in the PKC−/−:PKC+/− embryos were more severe than those observed in PKC+/−:PKC−/− embryos, suggesting that PKC plays a major role in preimplantation embryos. Because PKC and PKC have overlapping functions, the different requirements of the two PKC proteins probably reflected the differences in their expression levels. PKC−/−:PKC+/−, PKC+/−:PKC−/−, and PKC−/−:PKC−/− embryos failed to form a blastocoel at E3.5.

(G) Table summarizing the number of embryos obtained with each genotype by intercrossing PKC+/−:PKC+/− mice.

Figure S2. Distribution of Amot, Merlin, Kibra, and -Catenin, and the Distribution of Membrane Domain Proteins in Par1a/b DKD Embryos, Related to Figure 2 (A) Distributions of Amot and Yap between the one- to four-cell stages.

In the zygote (pronuclear stage), a faint Amot signal was observed in the plasma membrane. In the two-cell stage embryos, the Amot signal was stronger in the cell–cell contact sites than that in the noncontacting plasma membranes. In the four-cell stage embryos, the Amot signal was very faint. In the pre-compacting embryos shown here, the level of nuclear Yap was equivalent to or slightly higher than that of cytoplasmic Yap.

(B–D) Distributions of Merlin, Kibra, and -catenin in morula stage embryos. Note that Merlin, Kibra, and -catenin had uniform distribution throughout the plasma membrane (Kibra, Merlin) or AJs (-catenin).

(E) Reduction of Par1b protein in shPar1a/b-injected embryos. (F) Distributions of polarity marker proteins in Par1a/b DKD embryos. Yap was excluded from

the nuclei of the outer cells, whereas the distribution of PKC was not altered. Pard6b and E-cadherin were examined in the same embryos.

Figure S3. Amot Plays a Major Role in Activating the Hippo Pathway in Preimplantation Embryos, Related to Figure 3 (A) Absence of Amot proteins from Amot mutant embryos. (B) Knockdown of Amotl2 proteins in siAmotl2-injected embryos. Two siRNAs,

AMOTL2MSS226046 (siAmotl2-046) and AMOTL2MSS226047 (siAmotl2-047), were used. The latter exhibited slightly stronger KD effects and was used in most of the analyses.

(C) Yap and p-Yap distributions in Amotl2 KD embryos. Note that Amotl2 KD embryos had no apparent abnormalities.

(D) Normal distribution of aPKC in Amot-free embryos. (E) Projected images of an E4.5 Amot-free embryo that lacked the inner cells (type II). (F) Lats1/2 double mutant embryos that exhibited similar phenotypes to Amot-free embryos.

Left bottom: type I. Right: type II.

Figure S4. Interaction of Amot with Kibra, Merlin, Lats2, and F-Actin, Related to Figure 4 (A) Co-immunoprecipitation experiments showing the interactions of Amot with Kibra and

Merlin (Mer). (B) Co-immunoprecipitation experiments showing that Kibra interacts with the third PY motif

(PPEY) in Amot. (C) Co-immunoprecipitation experiments showing that Amot interacts with the N-terminal

domains in Kibra, which contains two WW domains. (D) Co-immunoprecipitation experiments showing the very weak interaction between Amot80

and Lats2. (E) F-actin polymerization and actin-binding activities of Amot proteins in HEK293T cells.

Exogenous Amot proteins were detected with the HA-tag.

Figure S5. Phosphorylation of S176 Controls the F-Actin Polymerization and Actin-Binding Activities of Amot Proteins in HEK293T Cells, Related to Figure 6 (A) F-actin polymerization and actin-binding activities of Amot-S176A/E proteins in HEK293T

cells. Exogenous Amot proteins were detected with the HA-tag. (B) Co-IP experiments showing the Merlin-dependent interaction between Amot-S176A/E and

E-cadherin. (C) Distribution of PKC proteins in Amot-(45–100)-expressing embryos.

Supplemental Experimental Procedures Embryo Culture and Manipulation One- or two-cell stage embryos were recovered from oviducts using standard protocols [1] and cultured as described previously [2]. The dissociation of 32-cell stage embryos was performed as described previously [3]. Generation of Protein Kinase C-Deficient Mice The targeting strategy is shown in Figure S1C. Mouse genomic DNA clones that included exons 6–10 of the PKC gene (Prkcz) were isolated from 129/SvJ genomic libraries (Stratagene). A 5.0-kb Aor51HI-XbaI fragment that included exons 6 and 7 and a 5.2-kb XbaI-XbaI fragment that included exons 8–10 were subcloned into the pBluescriptSK(+) vector, which contained a diphtheria toxin A gene. The loxP-flanked neomycin resistance gene (neo) was inserted into the XbaI site located in intron 7. The third loxP fragment was inserted into the BbeI site located in intron 9. The linearized vector was introduced into 129/SvJ-derived CCE embryonic stem cells by electroporation. To identify the homologous recombinant ES cells, genomic DNA from G418-resistant ES clones was digested with HindIII or ApaI and then hybridized with a 5′ or 3′ probe (Figure S1D). A targeted clone was injected into C57BL/6J blastocysts to generate chimeric mice. Male chimeric mice were bred with C57BL/6J mice. DNA from tail biopsies of the mice was analyzed to confirm germ-line transmission of the mutant allele by PCR using the following primer set: 5′-AAA GGG GCA CTG GAG ATT AAA CCC-3′ and 5′-GGA ATT ACC ACA CGA CCT AGC AGC-3′ (Figure S1E). The resulting Prkcz+/flox mice were bred with Actin-Cre C57BL/6J transgenic mice (a gift from Susan Dymecki) to generate heterozygous mutant (Prkcz+/−) mice. The Prkcz+/− mice without Cre were selected and interbred to generate homozygous (Prkcz−/−), Prkcz+/−, and wild-type (Prkcz+/+) mice for use in experiments. Plasmids Poly(A)-tailed RNAs were prepared from pcDNA3.1-poly(A)83 [4]-derived plasmids. pcDNA3.1-poly(A)83 containing -globin or Lats2 cDNA were described previously [5]. pcDNA3.1-poly(A)83 containing the dnPKC [6], Amot [7], or Amotl2 (MGC clone ID 5323888) cDNA were constructed by cloning the protein-coding regions of the respective mouse cDNAs, which were PCR-amplified from full-length cDNAs. The HA-tag was added to the N-terminus of Amot (including mutant forms), Amotl2, and Lats2. The identities of the PCR-amplified sequences were confirmed by DNA sequencing. Amot-CC was generated by deleting amino acid residues 410–670. Amot-PDZbd was generated by deleting the C-terminal four residues: 1, 120–1, and 123. Amot-mPY123 was generated by changing the three PY motifs (LPTY, PPEY, and PPEY) into FATE, GSEH, and GTEH, respectively. Amot-S176A and Amot-S176E were generated by changing S176 into A and E, respectively. Amot-(45–100) and Amot-(101–141) were generated by deleting amino acid residues 45–100 and 101–141, respectively. The rescue experiments used a concentration of 25 ng/l Amot RNA because injection of higher doses sometimes caused the abnormal aggregation of Amot proteins in the cytoplasm, which sequestered Yap from the nuclei without activating the Hippo pathway (data not shown).

shRNA plasmids validated by the RNAi Consortium were purchased from Sigma-Aldrich for gene knockdown. The ID numbers of the shRNA plasmids were as follows: shPard6b, TRCN0000054686; shPar1a, TRCN0000024105 and TRCN0000024106; and shPar1b, TRCN0000023985 and TRCN0000023987. Because of the limited availability of good antibodies, we only confirmed the knockdown of Par1b in Par1a/b double knockdown embryos (Figure S2E). However, we assumed that co-injection of shPar1a and shPar1b plasmids produced Par1a/b double knockdown embryos because injection of shPar1a or shPar1b did not cause any obvious abnormalities. We also confirmed that two shRNAs for Par1a/b had similar effects, suggesting

that the effects observed with shPar1a/b were specific to Par1a/b. The following plasmids were used for immunoprecipitation. pcDNA3-Myc-human

Merlin was a kind gift from Dr Georg Halder. pCMV-Tag4-Kibra, pCAG-Kibra-3xHA, and pCAG-Kibra-Flag were constructed by cloning the protein-coding region of Kibra cDNA [8] into the pCMV-Tag4 (Stratagene), pCAGGS-MCS-3xHA [9], and pCAGGS-MCS-Flag [9] vectors, respectively. pCAG-Kibra-WW1/2-Flag and pCAG-Kibra-C2-Flag lacked amino acid residues 1–94 and 704–784 of the Kibra protein, respectively. pMiw-Flag-Amot, pCAG-HA-Amot, pCAG-HA-Amot-mPY2, and pCAG-HA-Amot-mPY3 were constructed by cloning the protein-coding regions of Amot and mutant forms of Amot cDNAs into the pMiw [10] and pCAGGS [11] vectors. pCMV-Flag-Lats2 was generated by cloning the protein-coding region of Lats2 cDNA into the pCMV/SV-Flag vector. pCAG-E-cadherin-Flag and pCAG-E-cadherin-C-Flag were generated by replacing the EGFP-tag of the pCAG-E-cadherin-EGFP and -catenin of the pCAG-E-cadherin-C--catenin plasmids (gifts from Dr Nagafuchi) [12] with the Flag-tag, respectively. pCAG-HA-Merlin was constructed by cloning the protein-coding regions of mouse Merlin into the pCAGGS vector [11]. siRNAs Stealth RNAi siRNAs were purchased from Life Technologies for gene knockdown. The ID numbers of the siRNAs were as follows: siAmotl2, AMOTL2MSS226046 and AMOTL2MSS226047; siLats1, Lats1MSS275306, Lats1MSS275307, and Lats1MSS275308; and siLats2, Lats2MSS224891, Lats2MSS224892, and Lats2MSS224893. Both siRNAs produced efficient knockdown of Amotl2 and the most efficient, AMOTL2MSS226047, was used in most experiments. For Lats1 and Lats2, a mixture of three siRNAs was used for efficient knockdown to reduce the risk of off-target effects. Antibodies The following antibodies were used at the stated dilutions: rabbit polyclonal anti--catenin (C2206, Sigma-Aldrich), 1:4000; mouse monoclonal anti-Cdx2 (MU392-UC, BioGenex), 1:200; rat monoclonal anti-E-cadherin (ECCD-2) (M108, Takara Bio), 1:200; rabbit polyclonal anti-phospho-ERM (3141, Cell Signaling), 1:600; anti-HA tag (3F10) (11-867-423-001, Roche), 1:500; rabbit polyclonal anti-Lgl1 (sc-67244, Santa Cruz), 1:100; rabbit polyclonal anti-Par1b [13] and rabbit polyclonal anti-Pard6b (sc-67393, Santa Cruz), 1:50; rabbit polyclonal anti-PKC (sc-216, Santa Cruz), 1:100; rabbit polyclonal anti-Scribble (sc-28737, Santa Cruz), 1:200; rat monoclonal anti-Yap1 (H00010413-M01, Abnova), 1:2000; rabbit polyclonal anti-phospho-Yap (4911, Cell Signaling), 1:200; rabbit polyclonal anti-Nanog (RCAB0001P, Reprocell), 1:1000; and rabbit polyclonal anti-Gata6 (H-92 sc-9055, Santa Cruz), 1:100. Rabbit polyclonal anti-mouse Amot was raised against a peptide (STLERKTPIQILGQEPDAEMVEYLI) that corresponded to the C-terminus. The raised antiserum was affinity-purified and eluted with magnesium chloride. Rabbit polyclonal anti-phospho-Amot (S176) was raised against the phosphopeptide, C+HVRSLpSERLMQ, by Scrum Inc. (Tokyo, Japan). The antiserum was affinity-purified using the phosphorylated and nonphosphorylated peptides to obtain phosphopeptide-specific antibodies. Genotype Determination The genotyping of adults was performed by PCR using genomic DNA prepared from ear punches. For preimplantation embryos, the DNA was prepared after observing antibody-stained embryos. Genotyping was performed by PCR using primers that produced mutant and wild-type bands. The Amot alleles were genotyped using the following primers: Amot-Xf, TCA GCA GCA TCT TCT TTC GAG TCA G; Amot-Xr2, GAG TTG AAC TGA CTG ACC CTC AGG; and Amot-neo3, GTG GAT GTG GAA TGT GTG CGA G. A 400-bp band was amplified from the Amot mutant allele using Amot-Xr2 and Amot-neo3 primers. A 300-bp band was amplified from the Amot wild-type

allele using Amot-Xr2 and Amot-Xf primers. PKC alleles were genotyped using the following primers: Prkci wt FW, CCT CAC AAC AGC TCT GTG ACA TAG G; Prkci del FW, CGC ACA TAC ACA GAT GAG TGC C; and Prkci REV, CAA AGC CCT GCT CTC CTA GAG C. A 282-bp band was amplified from the PKC mutant allele using Prkci del and Prkci REV primers. A 361-bp band was amplified from the PKC wild-type allele using Prkci wt FW and Prkci REV primers. PKC alleles were genotyped using the following primers: Prkcz-1, AAA GGG GCA CTG GAG ATT AAA CCC; Prkcz-2, GGA ATT ACC ACA CGA CCT AGC AGC; and Prkcz-5, GCC GTG TGA AAT TGT GCT TCA GTG. A 322-bp band was amplified from the PKC mutant allele using the Prkcz-1 and Prkcz-2 primers. A 272-bp band was amplified from the PKC wild-type allele using the Prkcz-5 and Prkcz-2 primers. The PCR reaction conditions were as follows. For Amot, 95°C for 1 min, 40 cycles of 95°C for 30 s, 66°C for 30 s, 72°C for 1 min, and 72°C for 5 min. The PCR reaction conditions were as follows for PKC and PKC: 94°C for 3 min, 40 cycles of 94°C for 30 s, 66°C for 30 s, 72°C for 45 s, and 72°C for 3 min. Semiquantitative Determination of the Subcellular Distribution of Yap The subcellular distribution of Yap in the inner cells of Amot mutant embryos was determined semiquantitatively based on the Yap localization index (YLI): YLI = 1 indicated the clear exclusion of Yap from the nuclei; YLI = 2 indicated that the nuclear Yap level was slightly lower than the cytoplasmic Yap level; YLI = 3 indicated a nuclear Yap level similar to the cytoplasmic Yap level; YLI = 4 indicated a nuclear Yap level that was slightly higher than the cytoplasmic Yap level; and YLI = 5 indicated distinct nuclear Yap. An example of each YLI is shown in Figure 3F’. The mean YLI value for each embryo was used in the YLI analysis. In Figure 3F, each dot represents one embryo. Quantification of Immunofluorescent Signals The mean pixel intensities of Yap or p-Yap staining were measured using ImageJ. For Yap, we calculated the ratios of the mean intensity in the nuclei to the mean intensity in the cytoplasm. For each experiment, the values from single representative embryos were averaged and presented with the standard deviation. Similar results were obtained for several independent embryos. Phenotypic Classification of Amot RNA-Injected Amot-Free Embryos In the rescue experiments using Amot-free embryos by RNA injection, the phenotypes of the embryos were classified into four categories according to the following criteria: Rescue (N < C) = two or more of the inner cells with YLI = 1; No rescue (N > C) = two or more of the inner cells with YLI = 5; Constitutively active (CA) = two or more of the outer cells with YLI = 1 or 2; Weak rescue (N = C) = the embryos did not fit any of the three categories. Embryos with 16 cells or abnormal cells were excluded from the analysis. Calculation of the Frequencies of Embryos with Changes in the Marker Expression Pattern Par1a/b DKD caused developmental delay in a reproducible manner and were cultured for longer periods to adjust the cell numbers. Other manipulations, including Pard6b KD and Amot-free embryos, had no significant effects on cell proliferation. To estimate the frequency of embryos with the described patterns of marker expression in the Par1a/b DKD experiments (Figures 2F and S2F), we counted the number of embryos with abnormal Yap regulation as the denominator in order to clarify the relationship between the marker expression pattern and the abnormal Yap distribution pattern. To analyze the effects of Par1a/b DKD on the distribution of Yap, the total number of embryos was used as the denominator. A similar counting method was used for the analysis of the Amot distribution in dnPKC-expressing embryos (Figure S1B).

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