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Unique functions of Sonic hedgehog signaling during external genitalia development

¹ Center for Animal Resources and Development (CARD) and Graduate School of Molecular and Genomic Pharmacy, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan
² Program in Developmental Biology, The Hospital for Sick Children, and Department of Molecular and Medical Genetics, University of Toronto, Ontario M5G 1X8, Canada
³ Department of Molecular Neuropathology, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
⁴ Mammalian Genetics laboratory, National Institute of Genetics, Mishima, Japan
⁵ Western Regional Research Center, ARS, USDA, 800 Buchanan Street, Albany CA 94710, USA

View larger version (56K): [in a new window]   Fig. 1. Expression of Shh, Ptch1, Gli1, Gli2, Gli3, Bmp4 and Hoxd13 during external genitalia development. (Top) Embryonic GT (genital tubercle) development in mice; red regions indicate the urogenital sinus (left) and the GT (middle and right). (A,B) Shh is initially expressed in the epithelium of the outermost part of urogenital sinus at 10.5 dpc. (C,D) At 11.5 dpc, Shh expression is mainly localized to the distal region of the urethral epithelium. (E,F) Shh is broadly expressed in the urethral plate epithelium in the GT from 12.5 to 14.5 dpc. Ptch1 (G), Gli1 (H), Gli2 (I), Gli3 (J), Bmp4 (K) and Hoxd13 (L) are expressed in the mesenchyme of developing GT at 13.5 dpc. E,G-L are ventral views, B,D are transverse sections and F is a coronal section. Scale bars: 250 µm in A,C; 62.5 µm in B,D,F; 312.5 µm in E,G-L.

View larger version (103K): [in a new window]   Fig. 2. Effects of Shh protein and antibody administration on mesenchymal gene expression in the GT. GT explants were micro-dissected from 11.5 dpc embryos, implanted with Shh protein (s) and control BSA (b) beads, and cultured for 24 hours. (A-D) The expression of Ptch1 (A), Bmp4 (B), Hoxd13 (C) and Fgf10 (D) were augmented in regions adjacent to the Shh protein bead. Arrowheads and arrows indicate the augmented and endogenous expression, respectively. (E,F) Mesenchymal (endogenous) expression of Ptch1 in the GT explant was reduced after treatment with an anti-Shh neutralizing antibody for 24 hours. Scale bar: 250 µm in A-D; 312.5 µm in E,F.

View larger version (74K): [in a new window]   Fig. 3. Agenesis of GT in Shh-/- mice. (A,B) 12.5 dpc Shh-/- embryos show severe defects in the initiation of GT outgrowth (A, wild type; B, Shh-/- mice). Sagittal sections (as illustrated in a) were obtained and stained with Hematoxylin and Eosin. (D,F) Drastic reduction of Fgf8 expression in the GT of Shh-/- mice compared with wild type (C,E). Note that Fgf8 expression was detected in the limb buds, but was very reduced in the GT, of Shh-/- mice (D). Scale bars: 62.5 µm in A,B; 250 µm in C-F.

View larger version (39K): [in a new window]   Fig. 4. (A) The effect of inhibition of Shh activity by anti-Shh (5E1) antibody addition during GT formation. GTs from 12.5 dpc embryos (A) were cultured for 96 hours with 5E1 antibody (C) or with control antibody (B). The region above the line corresponds to GT (A). GT formation was retarded by 5E1 antibody application but not with control antibody. Reduced cell proliferation of anti-Shh antibody treated GTs is shown by a graph (D; control treated cell number is shown as 100%). Cultures of 11.5 dpc derived GTs showed more prominent reduction. Scale bar: 250 µm in A-C.

View larger version (72K): [in a new window]   Fig. 5. Aberrant ventral GT histogenesis induced by perturbing Shh signaling. Anti-Shh antibody addition induced ventral dysmorphogenesis (A, control section; B, 5E1 treated section which lacks midline urethral plate). Anti-Shh antibody mediated inhibition of mesenchymal Fgf10 gene expression in the ventral GT (coronal sections (C,D); C, control section; D, 5E1 treated section; arrowheads indicate the expression). GT phenotype of Gli2 mutants. Gli2-/- mice (16 dpc) display drastic abnormalities in the ventral GT formation phenocopying the Ab-treated specimen (E,F, coronal sections; E, wild type; F, Gli2-/- mutant). Aberrant ventral shape is marked by a line (B,F). Scale bars: 62.5 µm in A-D; 31.25 µm in E,F.

View larger version (50K): [in a new window]   Fig. 6. Apoptosis during normal GT development. (A) Sectioning of 10.5 dpc embryos revealed apoptotic cells in the epithelium of the outermost part of the urogenital sinus before GT outgrowth. (B) At 11.5 dpc, apoptotic cells were found in the urethral epithelium and mesenchyme in the distal region of the GT. The GT region is marked by a white line. (C) Apoptosis was mainly localized to the mesenchyme in the distal region adjacent to the urethral plate epithelium at 13.5 dpc. (A) Transverse section. Scale bars: 62.5 µm in A; 250 µm in B; 312.5 µm in C.

View larger version (72K): [in a new window]   Fig. 7. Increased cell death and decreased cell proliferation in Shh-/- mutant GT. (A-D) Apoptosis in 10.5 dpc (A,B) urogenital sinus and in 11.5 dpc (C,D) GT of wild type (A,C) and Shh-/- (B,D) embryos was examined by TUNEL analysis. Striking increase of apoptosis was found in the urethral epithelium of Shh-/- mutant (B,D) and the results are summarized in G. (E,F) Expression of Bmp4 in 10.5 dpc wild type (E) and Shh-/- (F) before GT outgrowth. In contrast to wild type, where expression is found only in the urogenital sinus mesenchyme, Bmp4 is expressed in the epithelium of Shh-/-. (H) Cell proliferation in 10.5 dpc urogenital sinus and 11.5 dpc GT of wild-type and Shh-/- embryos were determined by measuring BrdU incorporation. Shh-/- GTs showed a significant decrease in cell proliferation when compared with those of wild-type embryos. Six embryonic specimens were analyzed for wild-type and Shh mutants, and each sample was analyzed using four serial sections (G,H). (A-F) Transverse sections. Scale bar: 62.5 µm.

View larger version (25K): [in a new window]   Fig. 8. Model for the dual mode of Shh signaling in the developing GT. Red regions indicate endodermal regions shown as cross-sectioned circular regions (A, urogenital sinus at the outgrowth initiation period; B, urethral plate) and green lines represent ectoderm (A,B). Blue regions represent mesenchymal region and yellow portions for epithelia that normally express Shh (A, the epithelium of the outermost part of urogenital sinus at 10.5 dpc; B, the urethral plate epithelium emanating Shh at about 13 dpc). Altered expression of Fgf8 and Bmp4 are illustrated associated with the Shh mutation, which may account for the lack of GT outgrowth (A). Mesenchymal Fgf10 gene may be a candidate responding gene for the epithelial derived Shh (B).