7.2.3 Shh Signaling Pathway

Shh plays a dynamic role in the development of cloacal derivatives. Strong Shh expression can be detected throughout the endodermally derived cloacal epithelium before GT initiation, and its expression is later maintained in the urogenital sinus epithelium and the urethral epithelium. Shh signaling is essential for GT development. Global Shh knockout embryos demonstrate agenesis of the GT, along with a persistent cloaca.15,26 This phenotype is accompanied by a significant reduction or in some cases loss of genital-specific gene expression from both the urethral epithelium and the neighboring genital mesenchyme.15,26 Moreover, the requirement of Shh signaling extends beyond GT initiation. Using an inducible conditional knockout model, both Lin et al. and Seifert et al. independently showed that removal of Shh at later stages of GT outgrowth leads to moderate to mild GT underdevelopment with an open urethra (hypospadias).14,16

Shh exerts its growth promoting role, at least in part, through maintaining the GT signaling center dUE. In both straight and conditional Shh mutant embryos, expression of Fgf8, the best characterized marker for dUE, was diminished. More importantly, restoration of Fgf8 genetically by forced expression of a gain-of-function β-catenin allele in the urethral epithelium partially rescues the GT initiation defect and a subset of GT-specific gene expression.14,42 How exactly Shh maintains the dUE signaling center remains elusive. Existing evidence indicates that Shh does not act cell-autonomously on the urethral cells, as conditional deletion of its signal effector Smoothened in the urethral epithelium does not affect normal GT development.14,16 On the contrary, a series of tissue-specific knockout studies showed that the urethral epithelial-expressed Shh acts on the surrounding genital mesenchyme and the overlying genital ectoderm.14,16 Conditional ablation of Smoothened in the genital mesenchyme leads to a severe GT underdevelopment similar to that observed in global Shh knockouts.14 In a separate report, conditional knockout of Smoothened in the genital ectoderm was shown to be associated with a defect in urethral tube closure.16 However, this urethral phenotype does not seem to be as severe as in other Shh mutants. Taken together, it is more likely that Shh maintains dUE signaling through reciprocal interactions with the neighboring mesenchymal cells. BMP signaling might be an important target in this feedback loop. A simultaneous upregulation of Bmp4 expression, and a downregulation of its antagonist Noggin expression, was observed in the conditional Shh knockout embryos.14 Whether this alteration in Bmp pathway activity directly leads to the failure of dUE maintenance remains to be tested.

On the other hand, Shh also regulates GT and cloaca development independent of dUE signaling. Shh plays an essential role in patterning the cloacal field and this function cannot be duplicated by either WNT or Fgf.14 Even with Fgf signaling restored, the urethra remains trapped within the body in the absence of Shh, while the cloaca remains unseparated.14 The GT outgrowth in these embryos is stalled at around E12.5, indicating that a properly formed cloaca is a prerequisite for continued GT development. The failure of cloaca septation is also observed in the conditional Smoothened knockout animals, which further supports an obligatory requirement for Shh signaling in the PCM. Shh exerts its function in the PCM and GT mesenchyme by regulating the length of the cell cycle.17 In the absence of Shh signaling, the cell cycle extends from 8 h to 14.5 h, which leads to a stunning 75% reduction in growth. In addition, Shh also maintains the expression of Hoxa13 and Hoxd13 genes in the developing GT. The expression of these genes cannot be detected in the Shh knockout embryos even with Fgf signaling restored.14

It’s obvious that Shh plays a key role in regulating growth and patterning of both the limb and the GT. However, the expression pattern of Shh also marks the intriguing difference between the two types of appendages. Shh expression was only detected in the cloacal epithelium and later in the urethral epithelium of the GT, while in the limb bud, its expression was detected in the posterior limb mesenchyme.15,26,50 The function of Shh in the GT appears to be more complicated. This is because GT development and cloacal patterning are intrinsically coupled, and both processes are an integral part of caudal embryonic development. Experimental evidence so far suggests that Shh is a key factor that coordinates the whole process, as Shh expression can be detected days before GT development starts.