Multiphasic and tissue-specific roles of sonic hedgehog in cloacal septation and external genitalia development

Abstract

Malformations of the external genitalia are among the most common congenital anomalies in humans. The urogenital and anorectal sinuses develop from the embryonic cloaca, and the penis and clitoris develop from the genital tubercle. Within the genital tubercle, the endodermally derived urethral epithelium functions as an organizer and expresses sonic hedgehog (Shh). Shh knockout mice lack external genitalia and have a persistent cloaca. This identified an early requirement for Shh, but precluded analysis of its later role in the genital tubercle. We conducted temporally controlled deletions of Shh and report that Shh is required continuously through the onset of sexual differentiation. Shh function is divisible into two temporal phases; an anogenital phase, during which Shh regulates outgrowth and patterning of the genital tubercle and septation of the cloaca, and a later external genital phase, during which Shh regulates urethral tube closure. Disruption of Shh function during the anogenital phase causes coordinated anorectal and genitourinary malformations, whereas inactivation during the external genital phase causes hypospadias. Shh directs cloacal septation by promoting cell proliferation in adjacent urorectal septum mesenchyme. Additionally, conditional inactivation of smoothened in the genital ectoderm and cloacal/urethral endoderm shows that the ectoderm is a direct target of Shh and is required for urethral tube closure, highlighting a novel role for genital ectoderm in urethragenesis. Identification of the stages during which disruption of Shh results in either isolated or coordinated malformations of anorectal and external genital organs provides a new tool for investigating the etiology of anogenital malformations in humans.

Fig. 1.

Temporal dissection of Shh function in anogenital development. Shh was inactivated in Shh^creERT2/C mutants by administration of tamoxifen to pregnant females at the embryonic days shown along the top line. Hedgehog signaling was inactive 24 hours after tamoxifen treatment (confirmed by absence of Ptch1; data not shown). (A-C) Shh is required both for genital tubercle outgrowth and for cloacal septation from E10.5 to 12.5 (the anogenital phase). Loss of Shh signaling during this time window results in a persistent cloaca, truncated outgrowth and ventral hypoplasia of the genital tubercle. Earlier removal of Shh results in more severe outgrowth defects. (D-G) Shh signaling continues to be required during a second period, from E13.5 to 16.5 (the external genital phase), for normal urethragenesis and genital patterning. This phase follows perineum formation at E13.0, and mutants have external genital defects but separate anorectal and urogenital sinuses. (H,I) Loss of Shh signaling at early (H) and late (I) stages has similar effects on males and females.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.

Fig. 6.

Shh signals directly to genital ectoderm to maintain a closed urethral tube. Ventral views with distal towards the top in A,C-H; ventral is towards the bottom in B. (A,B) Genital tubercles showing Ptch1 expression at E14.5 (A) and E12.5 (B). Ptch1 transcripts are detectable in genital tubercle mesenchyme and in the ventral genital ectoderm (black arrows). The urethral epithelium is negative for Ptch1. (C,D) Control Smo^C/C without cre (C) and Msx2^cre;Smo^C/C (D) mouse genitalia at postnatal day 0 (P0). Arrowheads in D mark hypospadias. (E,F) Lysotracker Red staining at E16.5 shows that cell death is not increased along of the urethral seam (white arrowheads) of Msx2^cre;Smo^C/C embryo. Note ectopic cell death in the glans (F). (G,H) Deletion of Smo in the endoderm of Shh^GFPcre;Smo^C/C mutants (H) does not alter development of the perineum or urethral plate (compare H with control in G). UE, urethral epithelium.