Deletion of Shh during the anogenital phase results in a persistent cloaca and external genital malformation. Lineage mapping of Shh descendant cells in male Shh^creERT2/C;R26R embryos harvested at E18.5 following tamoxifen injections at E9.5-12.5. (A-C) Early (E10.5-12.5) loss of Shh signaling results in persistent cloaca and truncated genital outgrowth. (D) Deletion of Shh at E13.5 leads to incomplete closure of the preputial folds and hypospadias. (A′-C′) Sections of A-C. Septation of the cloaca is incomplete and the perineum is absent because Shh descendant cells fail to reach the cloacal membrane. (D′) Section through D. Note the severe proximal hypospadias and normal perineum. ARS, anorectal sinus; DS, dorsal swellings; G, glans; PER, perineum; PF, preputial folds; UGS, urogenital sinus; URS, urorectal septum.

Shh directs cloacal septation and genital tubercle outgrowth in a time-dependent manner. Analysis of anogenital and tail development in E14.5 embryos using OPT (A-D′), whole-mount X-gal staining (E-G) and histology (H-J). (A-D) Loss of Shh signaling in Shh^creERT2/C;R26R mice leads to a stage-specific shortening of the hindgut and urethral plate (A-C) compared with wild-type (Shh^GFPcre/+;R26R) mice (D). (A′-D′) High magnification images of specimens in A-D. (E-G) Loss of Shh signaling at E10.5 results in caudal truncation, loss of notochord and floorplate descendant cells, and loss of hindlimb digits (E), whereas longer exposure to Shh allows further development of caudal axis structures (F,G). Arrows (E,F) mark the posterior limit of midline Shh descendant cells. (H-J) Sagittal sections of the anogenital system shows that posterior expansion of urorectal septum mesoderm and growth of the urethral plate correlate with length of exposure to Shh. BL, bladder; GT, genital tubercle; HG, hindgut; UP, urethral plate; URS, urorectal septum.

Removal of Shh during the anogenital phase causes regionalized decreases in cell proliferation in urorectal septum mesenchyme. (A-E) BrdU labeling (A-D) and proliferative index (E) in anogenital region of control (Shh^+/C) and Shh^creERT2/C embryos. Shh signaling was inactivated at E11.5 (tamoxifen injection at E10.5) and embryos were examined at E12.5. Posterior is towards the left. Dotted lines in A and B outline the URSM. Yellow boxes in C and D indicate counting frames at posterior, dorsal and ventral positions of the URSM. (E) Comparison of the proliferative index at all three positions in Shh^+/C and Shh^creERT2/C embryos shows that the proliferative index is significantly lower in the posterior URSM of Shh^creERT2/C mutants (asterisk) but not significantly different at dorsal or ventral positions.

Deletion of Shh during the external genital phase leads to hypospadias. (A,B) Inactivation of Shh at E14.5 and 15.5 causes an ectopic opening of the urethra in the proximal penis (arrows). (A′,B′) Histological analysis of Shh^creERT2/C embryos shown in A,B shows a rupture of the ventral genital ectoderm. lacZ-positive urethral endoderm in a Shh^creERT2/C embryo (B′) shows the boundary between endoderm and ectoderm. (C,C′) Loss of Shh signaling at E16.5 did not affect ectodermal integrity.