Fig 1: Spatiotemporal patterns of Shh, Fgf10, and Fgfr2 expression during guinea pig external genital development. Whole-mount images are ventral (A–C,F–H,L–N) or dorsal (D,E) views of the developing external genitalia of guinea pigs (E,G) with the distal at the top, showing mRNA expression of Shh (A–E), Fgf10 (F–H) and Fgfr2 (L–N). Images in (I–K) are transverse sections of the developing penises of guinea pigs and mice, showing Fgf10 protein (in red; blue is Dapi) localization. Note that Shh mRNA is exclusively expressed in the urethral epithelium and prepuce during the later stages (A–E) of developing EG in guinea pigs. Fgf10 mRNA and protein mainly localize in the urethral epithelium at early stages in the guinea pig genital tubercle (GT) (F–I) and also in the preputial lamina (ppl) in E38 penis (J) of guinea pigs. However, in mouse GT, Fgf10 protein mainly localizes in the mesenchyme. Fgfr2 mRNA is mainly expressed in the urethral epithelium in guinea pig GT. Arrowheads in (D,E) point to the tiny Shh expression domain, while arrows in (H) indicate the Fgf10 expression domain in labioscrotal swellings. Abbrev: ppl and ue are the same as in Figure 1. Scale bars in (A–H,L–N): 500 µm; in (I–K): 100 µm.
Fig 2: In situ gene expression analysis of guinea pig genital tubercle (GT). Images are ventral (A–C,E,G,I,J,M,O,Q) or dorsal (D,F,H,K,L,N,P,R) views of E23-E23.5 guinea pig (excerpt for J, which is E22) GTs with the distal at the top. The tail has been removed from all embryos. Purple or blue staining in each image indicates the gene expression domain. Developmental stages are labeled upright in each image. Note that Shh is expressed in the urethral epithelium (A) and its receptor is expressed in the adjacent mesenchyme (B). Hoxd13 (C,D), Bmp4 (E,F), and Wnt5a (Q,R) are expressed in genital mesenchyme. Bmp7 is expressed in the distal urethral epithelium (very weak), genital mesenchyme, and developing mammary glands (G,H). Fgf8 expression is located in the urethral epithelium at E22 (J), but it is only weakly expressed in the distal tip part of the urethral epithelium and can only be seen in dorsal view at E23 (I,K,L). Fgf10 expression in genital mesenchyme is very weak and is relatively strong in urethral epithelium (M,N). Fgfr2 expression is mainly in the urethral epithelium (O,P). Abbrev: aer, apical ectodermal ridge; gt, genital tubercle; hl, hindlimb; mg, mammary gland; ue is the same as Figure 1. Scale bars: 500 µm.
Fig 3: The effect of Shh and Fgf-10 proteins on urethral groove and preputial development. Images in (A–L) are ventral views of mouse (A–F) and guinea pig (G–L) genital tubercles (GTs) with the distal end at the top. Broken line in images (D–F) shows the edge of urethral groove. Note that Hedgehog or Fgf inhibitors induce urethral groove formation but inhibit preputial development in cultured GTs of mice; the most significant effect is seen in cultured GTs with both inhibitors (D–F). Shh or Fgf-10 protein induces the formation of preputial swellings in cultured GTs of guinea pigs, and the most obvious results were found in both protein-treated GTs (J–L). Abbrev: uo, urethral opening; gp, ps, ss, and up are the same as in Figure 1, ug is the same as in Figure 2. Scale bars: 500 µm.
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