Wang S et al. (2013), Par6b regulates the dynamics of apicobasal pola...

XB-ART-47793

PLoS One 2013 Oct 08;810:e76854. doi: 10.1371/journal.pone.0076854.

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Par6b regulates the dynamics of apicobasal polarity during development of the stratified Xenopus epidermis.

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

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During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the "basolateral" state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.

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Species referenced: Xenopus laevis
Genes referenced: cadm1 cdh1 cdh3 crb3 ctnnb1 klf6 krt12.4 llgl2 lpar6 pard6b scrib sox2 tuba4b
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Phenotypes: Xla Wt + pard6b MO (Fig 4 A A^1 B B^1) [+]

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	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.

References [+] :

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