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First published online 1 November 2006
doi: 10.1242/dev.02621

Development 133, 4783-4792 (2006)
Published by The Company of Biologists 2006
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{Delta}Np63 plays an anti-apoptotic role in ventral bladder development

Wei Cheng1,2, W. Bradley Jacobs3, Jennifer J. R. Zhang1,2, Anne Moro1,2, Jin-Hyung Park1,2, Michelle Kushida1,2, Wei Qiu2, Alea A. Mills4 and Peter C. W. Kim1,2,*

1 Department of Surgery, Hospital for Sick Children, Toronto, M5G 1X8, Canada.
2 Program for Infection, Immunity, Injury and Repair, Hospital for Sick Children, Toronto, M5G 1X8, Canada.
3 Program of Developmental Biology, Hospital for Sick Children, Toronto, M5G 1X8, Canada.
4 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA.


Figure 1
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  		Fig. 1. BE in humans and p63-/- mice. (A) BE with separation of pubic bones and genitalia in a female. (B) Covered BE in a male. (C) The wild-type E18.5 embryo (10x). (D) BE in an E18.5 p63-/- embryo (10x). (E,F) Hematoxylin and Eosin staining of sagittal sections of wild-type (E) and p63-/- (F) E18.5 embryos (40x). White arrow: umbilical hernia. (G,H) Hematoxylin and Eosin staining of transverse sections of E18.5 wild-type (G) and p63-/- (H) embryo pelvises (40x). (Image A,B courtesy of J. L. Salle, Hospital for Sick Children, Toronto, Canada).

 

Figure 2
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  		Fig. 2. Ontogeny of p63 on sagittal sections of wild-type embryos (fluorescent immunohistochemistry, 100x). Sagittal sections of E11.5 embryos transect the epithelium tangentially at the distal UGS, accounting for the wider expression pattern at the distal urogenital sinus (A,E). (A-D) p63 (4A4) expression. (E-H) {Delta}Np63 isoform expression. (I-L) TAp63 isoform expression. Blood cells within the mesenchyme are autofluorescent. (M) RT-PCR of wild-type E15.5 bladder, using adenoviruses with {Delta}Np63, TAp63, p63{alpha}, p63ß and p63{gamma} constructs as controls. Arrows in A-J and M represent the positive immunoreactivities and RT-PCR bands of wild-type bladder samples. B, wild-type bladder cDNA; V, adenoviruses containing {Delta}Np63, TAp63, p63{alpha}, p63ß and p63{gamma} constructs.

 

Figure 3
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  		Fig. 3. p63 (4A4) expressions in E11.5 and E14.5 wild-type embryos. (A) p63 immunofluorescent staining of a sagittal section of an E11.5 embryo (20x). (B) p63 immunofluorescent staining of transverse sections of an E11.5 embryo pelvis (40x). (C) p63 immunofluorescent staining of an E11.5 UGS (200x). Arrows in A-C indicate the ventral aspects of the embryos. (D) Colorimetric immunostaining of a transverse section from an E14.5 embryo (20x). High-magnification view of ventral (E) and dorsal (F) skin of the E14.5 embryo (600x).

 

Figure 4
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  		Fig. 4. Ventral and dorsal epithelia of E18.5 wild-type and p63-/- bladders. (A-D) Hematoxylin and Eosin staining (600x). (E-H) Immunofluorescent staining of cytokeratin 18 (K18) (400x, confocal microscopy). (I-L) Immunofluorescent staining of uroplakin 3 (630x, confocal microscopy). (A,C,E,G,I,K) Wild type. (B,D,F,H,J,L) p63-/-. Arrows represent the epithelial layers.

 

Figure 5
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  		Fig. 5. (A,B) Hematoxylin and Eosin staining of the sagittal sections of E11.5 wild-type and p63-/- embryos (100x). (C,D) Fluorescent TUNEL staining (arrows) of wild-type and p63-/- UGS (200x). The DAPI staining of nuclei is shown in red to increase color contrast. (E,F) The colorimetric immunostaining (arrows) for cleaved caspase-3 in E11.5 wild-type and p63-/- UGS (200x). (G,H) Fluorescent TUNEL staining (arrows) of sagittal sections through the oral cavity of E11.5 wild-type and p63-/- embryos (400x). (I,J) The qPCR relative expressions of Bax and Apaf1 in E12.5 and E13.5 p63-/- bladders. (A,C,E,G) Wild type. (B,D,F,H) p63-/-. HG, hindgut.

 

Figure 6
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  		Fig. 6. The E13.5 p63-/--bladder primary cell cultures were transfected with adenoviruses expressing GFP, TAp63{gamma}, {Delta}Np63ß and {Delta}Np63{gamma}. The tensiometry of Bax, Apaf1 and ß-actin bands was recorded. The ratios of Bax/ß-actin and Apaf1/ß-actin of the specimens were compared with that of the GFP-infected controls (assigned to be 100). (A,B) Relative Bax/ß-actin (A) and Apaf1/ß-actin (B) ratios (Student's t-test, *P<0.05, **P<0.01). (C,D) Immunohistochemical staining of p53 in E11.5 wild-type (C) and p63-/- (D) UGS (200x). Arrows represent the ventral UGS. (E,F) Immunohistochemical staining of p73 in E11.5 wild-type (E) and p63-/- (F) UGS (100x). (G,H) The qPCR relative expressions of p53 (G) and p73 (H) in E12.5 and E13.5 wild-type (WT) and p63-/- bladders.

 

Figure 7
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  		Fig. 7. Epithelial-mesenchymal interactions. (A,B) Msx1 expressions (immunohistochemistry) in transverse sections of E14.5 wild-type (A) and p63-/- (B) bladders (100x). (C,D) Fgf8 in situ hybridization in the sagittal sections of E11.5 wild-type (C) and p63-/- (D) UGS (100x). (E,F) m-snail in situ hybridization of E14.5 wild-type (E) and p63-/- (F) bladders (sagittal sections) (100x). (G,H) BrdU incorporation in the sagittal sections of wild-type (G) and p63-/- (H) E11.5 UGS (100x). Arrows in A-H represent the ventral UGS. (I) Histogram of cell proliferation in both epithelium and mesenchyme of E11.5 wild-type and p63-/- UGS.

 

Figure 8
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  		Fig. 8. Bladder smooth-muscle development. (A,B) Smooth-muscle heavy-chain myosin expressions in the transverse sections of the E14.5 wild-type (A) and p63-/- (B) bladders. The intestinal muscular wall serves as an internal control (arrow, 100x). (C,D) Smooth-muscle {alpha}-actin actin expressions in the sagittal section of E18.5 wild-type (C) and p63-/- (D) bladders (40x). Large arrow in C,D represent the ventral bladder walls. Small arrow in D represents the dorsal bladder wall. (E,F) Hematoxylin and Eosin staining of E18.5 wild-type (E) and p63-/- (F) bladders (sagittal sections, 200x).

 

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