Genital tubercle development. (A-E) Schematics illustrating GT development. Preputial swellings grow ventrally around the GT (red arrows). Anogenital distance, perineum and scrotal swellings are larger in males (red asterisks). (F) Whole-mount β-galactosidase staining of ShhGFPCre;Rosa26RlacZ embryo shows lineage of Shh-expressing cells (blue) in epithelia lining the PHUR, PLUR, bladder, rectum, intestine, perineum, bulbourethral glands, preputial glands and UGS ridge. In males, the mesenchyme begins to septate the PHUR epithelium (red arrowheads in E,F).

Sexual differentiation of the external genitalia. (A-L) Whole-mount β-galactosidase staining of ShhGFPCre;Rosa26RlacZ embryos. Descendants of the Shh-expressing lineage (blue) contribute to preputial glands, epithelium of PHUR, perineum, rectum/intestine and hair follicles. (M-T) Schematics illustrate external genitalia. From E15.5-17.5 in males, mesenchyme grows distally, the urethra septates (red arrowheads), the proximal urethral meatus closes, the urethral seam forms and two right-angle bends develop in the urethra (red arrows). Male bulbourethral glands (black arrowheads), scrotal swelling/fold and perineum are larger. In both sexes, the preputial seam is seen as an indentation along the prepuce ventral midline (black asterisks in M,O). (U,V) H&E-stained external genitalia sections. (Q-V) At E17.5, the male urethra is patent along its length and open at the urethral meatus; in females, the urethral plate (upe) is present and the urethra opens at the proximal urethra meatus. (W-Y) Schematics illustrating adult male anatomy, showing the male urogenital mating protuberance (MUMP), corpora cavernosa (light purple), cartilage/bone (blue). Red arrowheads (in W) indicate position of cross-section (X). (Y) Two right-angle bends are present in the adult male urethra. The prostatic urethra (PRUR) is surrounded by prostate glands. Paired corpus cavernosum extend anteriorly to join the pubic bone (cc in Y).

Sexual differentiation of the reproductive ducts. Schematics illustrate 3D anatomy of reproductive ducts and their connection to the PLUR. At E15, the sexes are identical. Red arrows and associated text indicate sex-specific changes. Nephric and paramesonephric duct degeneration is complete by E17.5. (D) Section immunofluorescence of Hoxb7GFP embryo shows Hoxb7GFP⁺ ejaculatory ducts and seminal vesicles. Anterior prostate buds are seen (arrowheads). (F,G) The vagina grows in a posterior direction along the PLUR (red arrow), eventually separating from the PLUR and opening at the base of the clitoris (postnatally). (H) Section immunofluorescence shows differential expression in upper vagina (Pax2⁺) and SVB (Trp63⁺).

Sexual differentiation of the pelvic urethra. Schematics illustrate sagittal sections of bladder-PLUR junction at E17 (A,E) and adult (I,K). Sex differences (red asterisks) include: mesenchymal pad size/shape; thicker male lamina propria (lpplur) and thicker female muscle (muplur); muscle layer (muplur) continuous with detrusor in females (gap in males); and the bladder-PLUR (BL-PLUR) connection angle, close to 90° in males (I′), more linear in females (K′). (B,F) Whole-mount immunolabelling shows epithelial structures (Cdh1⁺). (C,G) Immunofluorescence of sagittal sections shows smooth muscle (green) and Upk (red) expression. Upk marks the bladder urothelium boundary (dotted arrows). (D,H) 3D illustrations of PLUR epithelium at birth (purple) shows prostate, ventral epithelial and urethral gland bud location; lateral (black), dorsal (white) and ventral (green) prostate glands and ventral epithelial buds (veb, blue) are outlined. (D′,H′) Underside views of ventral PLUR. (J) Cross-section through adult male PRUR shows labelling of epithelia (red), smooth muscle (green) and striated muscle (yellow), which marks the rhabdosphincter. Blood vessels (Acta2⁺, red arrowheads) and prostate glands (Cdh1⁺, white arrowheads) are seen. (K-N) In female adults, the vagina has elongated and a rhabdosphincter develops. (L-N) Sagittal BL-PLUR sections labelled with Upk (bladder urothelium) shows the adult BL-PLUR transition zone (dotted arrows). Urethral glands (arrowheads, L-N) are located close to the BL-PLUR boundary.

Morphology of the bladder fundus, neck, trigone and ureters. (A-E) Schematics illustrating location and morphology of the bladder regions, ureter and urethra connections at E15. Trigone and BL-PLUR boundaries are shown (red/black dotted lines). The bladder neck is more elongated in the adult (see Fig. 5M). (F) As the bladder grows, trigone morphology changes from an equilateral to an isosceles triangle. (G-L) Immunofluorescence of vibratome sections and whole-mounts of E18 ureter (G-I), adult ureter (J) and trigone (K,L), with labelling of urothelium (Upk), ureter muscle layer and detrusor muscle (Acta2) and Hoxb7-GFP in ureter urothelium (K). Approximate trigone boundary is shown (red dotted lines in I,K,L).

Radial patterning of the bladder mesenchyme activates and maintains smooth muscle differentiation. (A-D) Schematics illustrate mesenchymal and smooth muscle layers. The urothelium expresses Shh, signalling the mesenchyme to express Bmp4, Ptch1 and Gli1-3. Relative gene expression (lower-higher) and presence/absence of smooth muscle cells are shown. Muscle differentiation begins at the fundus and progresses towards the PLUR (B,C). (E,F) β-galactosidase-labelled Gli1-lacZ tissue sections show differential Gli1 expression in the UGS and bladder mesenchyme. (G,H) Immunolabelled bladder sections show urothelium (Upk) and smooth muscle (Acta2). (I,J) β-galactosidase-labelled Gli1-lacZ tissue sections show location of Gli1 and Laminin. Expression of Gli1 is strongest in the suburothelial lamina propria (sulpbl) at E14 (I) and E16 (J) and weak or absent in the outer lamina propria (olpbl). The sulpbl also contains numerous blood vessels (Laminin⁺, white arrowheads, I,J). (K) Immunolabelled vibratome sections show urothelium (Upk) and detrusor muscle (Acta2). Bright field (on the right) shows the convoluted adult bladder urothelium.

Protein expression defines the bladder urothelium and PLUR epithelium cell types. (A-F) Schematics illustrate epithelial layers; urothelium (red) and caudal UGS (CUGS)/PLUR epithelium (blue). Cross-sections below each panel illustrate location and gene expression of urothelial cell types. (D-F) From E14, bladder urothelium is divided into superficial (S), intermediate (I) and basal (B) cell layers. (G-N) Immunolabelling of LUT tissues. Expression of Trp63 is stronger in CUGS at E12 (G). Krt5 is not expressed in the bladder until after E14; however, expression is seen in the dorsal PLUR epithelium at E13 (H,I), which also shows stronger Trp63 expression. The PLUR superficial cell layer does not express Trp63 at E14 (J,K). (L-N) In the E16 (L,M) and E18 (N) urothelium, Upk is expressed by superficial (SC) and intermediate (IC) cells, Trp63 by ICs and Krt5-basal cells (BCs), and Krt5 exclusively by Krt5-BCs; however, these cells are found in both the basal and intermediate layers. By birth, SCs (or umbrella cells) are large and multi-nucleated (N). Up2-Cfp reporter mice show Upk-expressing cells (green, N). (O-X) Expression patterns in PLUR versus bladder regions (trigone, red dotted lines/arrows). (O) From E15, the PLUR epithelium is subdivided into superficial (S), intermediate (I) and basal (B) cell layers (S), which are thicker than the bladder urothelium. (P) Schematic shows bladder fundus/ventral neck (yellow), bladder dorsal neck/trigone (red) and PLUR epithelium (grey). (Q) All bladder regions express Upk. (R) Krt14 expressed in bladder neck/trigone and PLUR at E17. (S,U-X) Immunolabelling in the BL-PLUR. (S) Krt14, Krt5 and Trp63 expression. (T) Summary of BL-PLUR expression profiles in the cell layers from E17 to adult (TS15-28). White circles indicate differences between bladder regions; black circles indicate differences between PLUR and bladder. Krt20 is expressed by mature SCs of the bladder from E18 to adult (blue ‘+’ in T). (U) Foxa2 is expressed in bladder neck/trigone (U) and PLUR, but absent in the bladder fundus (data not shown). (V) E17 BL-PLUR showing tissue regions, Upk (green arrows) and Krt13 (red arrows) expression borders, with BL-PLUR transition zone in between (white arrows). (W,X) Adult PLUR showing Krt14, Krt5 and Krt13 expression in the epithelial layers.