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First published online 3 January 2007
doi: 10.1242/dev.02736

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Molecular analysis of coordinated bladder and urogenital organ formation by Hedgehog signaling

¹ Center for Animal Resources and Development (CARD), Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.
² Molecular Neuropathology Group, Brain Research Institute, RIKEN, Wako, Saitama, Japan.
³ RIKEN Center for Developmental Biology, Chuo-ku, Kobe 650-0047, Japan.
⁴ Department of Pediatrics and Program in Human Molecular Biology and Genetics, University of Utah, UT 84112, USA.

View larger version (76K): [in this window] [in a new window]   Fig. 1. Urogenital tissue formation in mice. (A-F) Histogenesis of the developing cloaca, GT, UP and bladder at E10.5 (A,D), E11.5 (B,E), E13.5 (C,F). H&E stained mid-sagittal sections of the cloaca, GT and bladder primordia are shown in D-F. b, bladder; bw, body wall; c, cloaca; gt, genital tubercle; pcm, peri-cloacal mesenchyme; t, tail bud; u, internal urethra; uc, umbilical cord; up, urethral plate.

View larger version (97K): [in this window] [in a new window]   Fig. 2. Expression patterns of Shh signaling genes in developing urogenital tissues. Shh is expressed in the cloacal epithelium, internal urethra, bladder and urethral plate (A-C). Serial tissue sections reveal Ptc1 (D-F) and Gli1 (G-I) expression in the adjacent mesenchyme. At E10.5, both Ptc1 (D) and Gli1 (G) are expressed in the PCM (yellow box). At E11.5 and E13.5, both genes are expressed in the mesenchyme surrounding the urethra, bladder and urethral plate (E,F,H,I). (J) Schematic depicting urogenital organs adjacent to the limb and the tail at E13.5. Shh-expressing epithelia are shown in red. b, bladder; bw, body wall; c, cloaca; cm, cloacal membrane (the ventral side of the cloaca composed by inner endodermal epithelia with surface ectoderm); gt, genital tubercle; hg, hindgut; pcm, peri-cloacal mesenchyme; r, rectum; u, internal urethra; uc, umbilical cord; up, urethral plate.

View larger version (67K): [in this window] [in a new window]   Fig. 3. Dysgenesis of the body wall, bladder and GT in Shh null mutants. (A-H) H&E-stained mid-sagittal sections of wild-type (A,B,E,F) and Shh-/- (C,D,G,H) specimens at E13.5 (A-D) and at newborn stages (E-H). Higher magnifications of the GT and bladder primordia (boxed in A,C,E,G) are shown in B,D,F,H. The blue arrows in D and H indicate mutant bladder primordia; the green arrows in E indicate internal (pelvic) urethra. am, abdominal muscle; b, bladder; bs, bladder smooth muscle; bw, body wall; eg, external genitalia; p, pelvic bone; r, rectum; u, internal urethra; uc, umbilical cord.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Urogenital tissue defects in Gli mutant mice. (A-C) Mid-sagittal H&E sections of urogenital organs in newborns with genotypes indicated. Urogenital tissue defects present in Gli2-/- embryos (B) were exacerbated in Gli1-/-;Gli2+/- embryos (C) as compared with controls (Gli1-/- or Gli2+/- embryos, data not shown). am, abdominal muscle; b, bladder; eg, external genitalia; p, pelvic bone; r, rectum; u, internal urethra.

View larger version (90K): [in this window] [in a new window]   Fig. 5. The del5-LacZ transgenic reporter allele detects active HH signaling in vivo. (A) A schematic diagram of the del5-LacZ DNA construct to generate transgenic mice. The promoter contains eight consensus Gli-binding motifs (red ovals) and a basal gamma-crystalline promoter upstream of the LacZ gene. (B-D) LacZ staining was detected in the PCM at E10.5 (A; yellow box and inset) and at subsequent stages in the PCM, urethral plate and bladder (C,D). Note that at E12.5 and E13.5, there is robust LacZ activity in bladder (white arrow) but not in the GT mesenchyme (black arrow). b, bladder; c, cloaca; gt, genital tubercle; u, internal urethra; uc, umbilical cord; up, urethral plate.

View larger version (82K): [in this window] [in a new window]   Fig. 6. HH-reporter expression in Shh null and Xt/Xt (Gli3) mutant embryos. (A-D) Expression of the del5-LacZ transgene in the limbs and in the neural tube of wild-type (A,C), Xt/Xt (B) and Shh null (D) embryos at E11.5. The expression level of del5-LacZ was reduced in Shh-/- (D) and augmented in Xt/Xt (B) mutant embryos (black arrows).

View larger version (98K): [in this window] [in a new window]   Fig. 7. Shh null mutants have reduced HH signaling in the developing urogenital organs. A reduction in LacZ activity from the del5-LacZ reporter of Shh-/- mutant PCM and bladder (B,D) was observed when compared with the control specimen (A,C). (C,D) Higher magnifications of the boxed areas show the bladder primordia. b, bladder; c, cloaca; gt, genital tubercle; pcm, peri-cloacal mesenchyme; u, internal urethra; uc, umbilical cord.

View larger version (45K): [in this window] [in a new window]   Fig. 8. Contribution to HH-responding bladder and external genitalia mesenchymes from the PCM. Above is a schematic depicting the inducible Gli1-CreERT2 allele and the experimental timecourse employed to labeling HH-responsive tissues. (A-D,F,G) LacZ staining of urogenital tissues in Gli1-CreERT2;R26R embryos that were administered tamoxifen at E8.759.0. (A,C) Whole-mount view of stained embryos at E10.5 and E13.5. (B,D) Mid-sagittal sections show the contribution of HH-responsive cells initially to the PCM (yellow box and inset) and to the mesenchyme of the GT and bladder (D, red arrow). (E) Diagram of newborn sagittal specimens; yellow boxes indicate likely primordia of penile bone and corporal bodies and the bladder, labeled and shown in F and G, respectively. b, bladder; c, cloaca; gt, genital tubercle; hg, hindgut; pcm, peri-cloacal mesenchyme; r, rectum; t, tail bud.

View larger version (61K): [in this window] [in a new window]   Fig. 9. Marker analyses and fate mapping of tissues responding to HH signaling in Shh mutants. (A-J) Tamoxifen-induced Cre labeling with the same experimental conditions as those described in Fig. 8. (A-D) E14.5 specimens were co-stained for LacZ activity (green) and immunohistochemically stained for differentiation markers (brown). (A,B) In the bladder (but not in the GT) LacZ labeled mesenchyme co-stained with anti-smooth muscle myosin (SMM). (C,D) The LacZ-positive dorsal GT mesenchyme overlapped with the tissue expressing AR. (E-J) In Shh mutants, reduced LacZ staining was detected in the embryonic GT and bladder mesenchyme (compare E with H, and F,G with I,J). (K) A schematic depicting the distribution of HH responding tissues at E9.5-10.5 and E13.5. b, bladder; bw, body wall; c, cloaca; cm, cloacal membrane (the ventral side of the cloaca composed by inner endodermal epithelia with surface ectoderm); gt, genital tubercle; hg, hindgut; pcm, peri-cloacal mesenchyme; up, urethral plate.

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