Wang S , Cha SW , Zorn AM , Wylie C .

R01HD044764 NICHD NIH HHS , R01 HD044764 NICHD NIH HHS

Xla Wt + pard6b MO (Fig3 B B^1 C, C^1)
Xla Wt + pard6b MO (Fig 4 A A^1 B B^1)
Xla Wt + pard6b MO (Fig 4 C C^1)
Xla Wt + pard6b MO (Fig 4 E)
Xla Wt + pard6b MO (Fig 4 E^1)
Xla Wt + pard6b MO (Fig 4.H)
Xla Wt + pard6b MO (fig.5.a)
Xla Wt + pard6b MO (fig.5.a)
Xla Wt + pard6b MO (fig.5.a)
Xla Wt + pard6b MO (fig.6.a,c)
Xla Wt + pard6b MO (Fig 6 D)
Xla Wt + pard6b MO (fig.6.d)
Xla Wt + pard6b MO (Fig 6 E)
Xla Wt + pard6b MO (fig.6.e)
Xla Wt + pard6b MO (fig.6.f)
Xla Wt + pard6b MO (fig.6.f)
Xla Wt + pard6b MO (fig.S3.a)
Xla Wt + pard6b MO (fig.S3.b)
Xla Wt + pard6b MO (fig.S3.c)
Xla Wt + pard6b MO (fig.S3.d)
Xla Wt + pard6b MO (fig.S3.f)

	Figure 1. The dynamics of Crb3 and Lgl2 subcellular localization in the developing Xenopus stratified epidermis. (A, B, C) Schematic representations of the epidermis development at blastula (st9), gastrula and neurula (st17) stages, respectively. Blue arrowheads in panel B illustrate the radial interdigitation of deep cells in the gastrula ectoderm. The black boxes mark the imaging area with the apical surface facing up. (D, F, G, H) Crb3-GFP (green) distribution in st9, 10.5, 12, 17 ectodermal cells, respectively. Red arrowheads indicate dynamic subcellular changes of Crb3-GFP. (E, I) GFP-Lgl2 (white) distribution in st9 and st17 ectodermal cells. (A–I) Double-headed black arrows on the left side of each panel show the boundaries of the superficial and deep layers. (D–I) Scale bars, 50 µm. doi:10.1371/journal.pone.0076854.g001
	Figure 2. The expression pattern of par6b mRNA and the subcellular localization of Par6b protein during development. (A) Sagittal view of par6b expression pattern in a st9 blastula embryo by whole mount ISH. (B) Sense par6b probe control in a st 9 embryo. (C) Transverse view of par6b expression pattern in a st 17 neurula embryo. (D) par6b expression pattern in a transverse section of st17 embryo. (E) Staining of E-cadherin in the epidermis in a transverse section of st17 embryo. (I–N) Staining with anti-HA of Par6b (red) and anti-β-catenin (green) antibodies on st9 or st17 ectoderm. (D–N). Double-headed arrows (black in panel D and E, white in panel I–N) respectively indicate boundaries of the superficial and deep layers. Scale bars, 50 µm. doi:10.1371/journal.pone.0076854.g002
	Figure 3. Par6b [pard6b] depletion causes epidermal cell dissociation. (A, A’) st32 uninjected embryos and magnified image of the epidermis. (B, B’) st32 embryos injected with Par6b-MO into the two animal ventral blastomeres at the 8-cell stage and corresponding magnified image of the epidermis. (C, C’) st32 embryos were injected with Par6b-MO and GFP mRNA into two animal ventral blastomeres at the 8-cell stage. Par6b-MO injected (GFP positive) epidermal cells were shed into petri dish. (D–F) 5 ng of Par6b-MO was co-injected with GFP into one animal ventral blastomere at 16-cell stage. Whole-amount staining of activated-Caspase 3 (Red), nucleus (white) and GFP (Green) indicated that the number of Activated-Caspase 3 positive cells was not increased in Par6b-depleted clones (GFP positive) at st30. doi:10.1371/journal.pone.0076854.g003
	Figure 4. Par6b depletion reduces epidermal cadherins. (A–C’) Embryos were injected with Par6b-MO together with GFP mRNA into one animal ventral blastomere at the 8-cell stage. Staining of E-cadherin (A, A’) or C-cadherin (C, C’) (red) and GFP (green) in sections of st15 embryos. Surface magnified view of whole-mount staining of E-cadherin and GFP of st15 injected embryos (B, B’). (D–F’) Phenotypes of uninjected, Par6b-MO injected alone, and Par6b-MO with Xt par6b mRNA injected embryos at st32 and st39. (G, H, I) Corresponding whole-mount staining of E-cadherin on st30 epidermis. doi:10.1371/journal.pone.0076854.g004
	Figure 5. Par6b depletion reduces protein levels but not the mRNA levels of E- and C-cadherin. (A) Western blot analysis of total protein extracts from uninjected, 20 ng and 40 ng Par6b-MO injected embryos with anti-C-cadherin and E-cadherin antibodies. (B) Quantitative RT-PCR results of e-cadherin and c-cadherin mRNA levels by two doses of Par6b-MO injection at st9, 12, 16 and 19. doi:10.1371/journal.pone.0076854.g005
	Figure 6. Par6b depletion stabilizes Crb3 on deep cell membranes, and destabilizes Lgl2 in both epidermal layers. (A) Crb3-GFP signal in a transverse section of st17 embryos. 25 ng of Par6b-MO was injected into one blastomere at the 2-cell stage. Left half is the uninjected side and right half is the Par6b-MO injected side. (B) Crb3-GFP distribution in uninjected epidermis, left box in figure A. (C) Crb3-GFP distribution in Par6b-depleted side, right box in figure A. (D, E) GFP-Lgl2 signal in transverse section of st12.5 and st17 injected embryos. 10 ng of Par6b-MO with RIDX was injected into one animal ventral blastomere at the 8-cell stage. (E) Western blot of total protein extracts from st12.5 and st16/17 Crb3-GFP or GFP-Lgl2 injected host transfer embryos with or without injection of 50 ng Par6b-MO. doi:10.1371/journal.pone.0076854.g006
	Figure S1. The expression pattern of par6b during development. (A) Sagittal view of par6b in a st IV oocyte by whole-mount ISH. (B, B') par6b expression pattern on a sagittal section of st9 embryo and the magnified view of the box area. (C) Lateral view of par6b expression pattern at st11. (D, D') par6b expression pattern on a sagittal section of st11 embryo and the magnified view of the box area. (E) Lateral view (head toward the left) of par6b expression pattern at st24. (F, F') par6b expression pattern on a sagittal section of st24 embryo and the magnified view of the box area.
	Figure S2. Control-MO injection does not change E- and C-cadherin expression. (A-B′) Embryos were injected with Par6b-MO together with GFP into one animal ventral blastomere at the 8-cell stage. Staining of E-cadherin (A, A′) or C-cadherin (C, C′) (red) and GFP (green) on the section of st17 injected embryos.
	Figure S3. Par6b depletion causes reduction of other epidermal adhesion molecules without an elevation of apoptosis. (A-F) Staining of β-, α- and γ- catenins, β1- and α5-integrins, and tight junction ZO-1 (red) respectively on sections of st17 epidermis with Par6b-MO injected clones (GFP positive, green). Scale bars, 50 um.
	Figure S4. Par6b depletion does not change ectoderm cell fate. (A) Quantitative RT-PCR assays of expression of ectodermal markers epidermal keratin, sox2 and n-cadherin mRNA levels after two doses of Par6b-MO injection from the late blastula (st9) to neurula (st21) stage. (B) Cytokeratin staining on the transverse section of st17 embryos that contain Par6b-MO injected clones (GFP positive). Scale bars, 50 um.
	Figure 7. Summary model of the role of Par6b in developing stratified Xenopus epidermis.(A) The blastula presumptive epidermis. Crb3 is all around membranes of both superficial and deep cells. Lgl2 is basolaterally restricted in superficial cells and evenly distributed on all membranes of deep cells. (B) The neurula differentiated epidermis. Crb3 is apically restricted in superficial cells whilst forms cytoplasmic punctae in deep cells. Lgl2 is basolaterally restricted in superficial cells and on all membranes of deep cells. E-cadherin has similar distribution pattern as Lgl2. (C) The Par6b-depleted neurula epidermis. Superficial cells have Crb3 on apical membrane but less Lgl2 on basolateral membrane. Deep cells have both Crb3 and Lgl2 evenly distributed all around the membrane. E-cadherin is lost in both layers. (A–C) Double-headed arrows on the right side of each panal indicate boundaries of the superficial and deep layers.

Billett, Fine structural changes in the differentiating epidermis of Xenopus laevis embryos. 1971, Pubmed, Xenbase

Billett, Fine structural changes in the differentiating epidermis of Xenopus laevis embryos. 1971, Pubmed , Xenbase
Bowerman, The maternal par genes and the segregation of cell fate specification activities in early Caenorhabditis elegans embryos. 1997, Pubmed
Cha, Wnt5a and Wnt11 interact in a maternal Dkk1-regulated fashion to activate both canonical and non-canonical signaling in Xenopus axis formation. 2008, Pubmed , Xenbase
Chalmers, Oriented cell divisions asymmetrically segregate aPKC and generate cell fate diversity in the early Xenopus embryo. 2003, Pubmed , Xenbase
Chalmers, aPKC, Crumbs3 and Lgl2 control apicobasal polarity in early vertebrate development. 2005, Pubmed , Xenbase
Chalmers, Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation. 2002, Pubmed , Xenbase
Cline, Characterization of mammalian Par 6 as a dual-location protein. 2007, Pubmed
Cunliffe, PAR6B is required for tight junction formation and activated PKCζ localization in breast cancer. 2012, Pubmed
Gao, Isoforms of the polarity protein par6 have distinct functions. 2004, Pubmed
Guo, Molecular genetics of asymmetric cleavage in the early Caenorhabditis elegans embryo. 1996, Pubmed
Hayase, The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells. 2013, Pubmed
Helfrich, Role of aPKC isoforms and their binding partners Par3 and Par6 in epidermal barrier formation. 2007, Pubmed
Hung, PAR-6 is a conserved PDZ domain-containing protein that colocalizes with PAR-3 in Caenorhabditis elegans embryos. 1999, Pubmed
Huynh, Bazooka and PAR-6 are required with PAR-1 for the maintenance of oocyte fate in Drosophila. 2001, Pubmed
Jamrich, Cell-type-specific expression of epidermal cytokeratin genes during gastrulation of Xenopus laevis. 1987, Pubmed , Xenbase
Joberty, The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. 2000, Pubmed
Keller, The cellular basis of epiboly: an SEM study of deep-cell rearrangement during gastrulation in Xenopus laevis. 1980, Pubmed , Xenbase
Kemphues, Identification of genes required for cytoplasmic localization in early C. elegans embryos. 1988, Pubmed
Kishi, Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm. 2000, Pubmed , Xenbase
Lee, EphrinB1 controls cell-cell junctions through the Par polarity complex. 2008, Pubmed , Xenbase
Lemmers, CRB3 binds directly to Par6 and regulates the morphogenesis of the tight junctions in mammalian epithelial cells. 2004, Pubmed
Levi, The distribution of E-cadherin during Xenopus laevis development. 1991, Pubmed , Xenbase
Levi, EP-cadherin in muscles and epithelia of Xenopus laevis embryos. 1991, Pubmed , Xenbase
Longo, Multicellular computer simulation of morphogenesis: blastocoel roof thinning and matrix assembly in Xenopus laevis. 2004, Pubmed , Xenbase
Margolis, Apicobasal polarity complexes. 2005, Pubmed
Martin-Belmonte, Epithelial cell polarity, stem cells and cancer. 2011, Pubmed
Mir, FoxI1e activates ectoderm formation and controls cell position in the Xenopus blastula. 2007, Pubmed , Xenbase
Mir, How the mother can help: studying maternal Wnt signaling by anti-sense-mediated depletion of maternal mRNAs and the host transfer technique. 2008, Pubmed , Xenbase
Müller, Molecular networks controlling epithelial cell polarity in development. 2003, Pubmed , Xenbase
Munson, Regulation of neurocoel morphogenesis by Pard6 gamma b. 2008, Pubmed
Muroyama, Polarity and stratification of the epidermis. 2012, Pubmed
Müsch, Mammalian homolog of Drosophila tumor suppressor lethal (2) giant larvae interacts with basolateral exocytic machinery in Madin-Darby canine kidney cells. 2002, Pubmed
Muthuswamy, Cell polarity as a regulator of cancer cell behavior plasticity. 2012, Pubmed
Nandadasa, N- and E-cadherins in Xenopus are specifically required in the neural and non-neural ectoderm, respectively, for F-actin assembly and morphogenetic movements. 2009, Pubmed , Xenbase
Petronczki, DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. 2001, Pubmed
Pieczynski, Protein complexes that control renal epithelial polarity. 2011, Pubmed
Reischauer, Lgl2 executes its function as a tumor suppressor by regulating ErbB signaling in the zebrafish epidermis. 2009, Pubmed
Rosse, PKC and the control of localized signal dynamics. 2010, Pubmed
Royer, Epithelial cell polarity: a major gatekeeper against cancer? 2011, Pubmed
St Johnston, Epithelial polarity and morphogenesis. 2011, Pubmed
Suzuki, The PAR-aPKC system: lessons in polarity. 2006, Pubmed
Tadjuidje, The functions of maternal Dishevelled 2 and 3 in the early Xenopus embryo. 2011, Pubmed , Xenbase
Tanentzapf, Interactions between the crumbs, lethal giant larvae and bazooka pathways in epithelial polarization. 2003, Pubmed
Tepass, crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. 1990, Pubmed
Torpey, Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos. 1992, Pubmed , Xenbase
Totong, PAR-6 is required for junction formation but not apicobasal polarization in C. elegans embryonic epithelial cells. 2007, Pubmed
Wang, Tight junction protein Par6 interacts with an evolutionarily conserved region in the amino terminus of PALS1/stardust. 2004, Pubmed
Watts, par-6, a gene involved in the establishment of asymmetry in early C. elegans embryos, mediates the asymmetric localization of PAR-3. 1996, Pubmed
Wodarz, Expression of crumbs confers apical character on plasma membrane domains of ectodermal epithelia of Drosophila. 1995, Pubmed
Yamanaka, Mammalian Lgl forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity. 2003, Pubmed