Twenty-five years ago, it was shown that proximodistal development of the external genitalia is dependent on the epithelial component of the genital tubercle. Removal of the epithelium from rat genital tubercles resulted in stage-dependent truncation of the baculum (penian bone) and erectile tissues (Murakami and Mizuno, 1986). Although these manipulations were done at relatively late stages of outgrowth, the observation was reminiscent of the permissive role of the apical ectodermal ridge in the limb bud and suggested that conserved mechanisms could be responsible for maintenance of outgrowth of both structures.
Fgf8 Is Not Involved in Genital Outgrowth
More recently, it was shown that Fgf8 is expressed in the distal tip of the urethral epithelium in a pattern that bears a striking resemblance to its expression in the AER at the distal tip of the limb bud (Haraguchi et al., 2000; Perriton et al., 2002). Surprisingly, genetic deletion of Fgf8 in the genital tubercle has no effect on external genital development (Seifert et al., 2009b). Indeed, analysis of Fgf8 target genes and the distribution of Fgf8 protein showed that Fgf8 is not even translated in the urethral epithelium (Seifert et al., 2009b). Although previous in vitro experiments had suggested a role for Fgf8 in the genital tubercle (Haraguchi et al., 2001), genetic studies in vivo showed that Fgf8 is not involved in genital development (Seifert et al., 2009b). Additionally, the other AER-associated Fgfs either are not expressed at any stage of genital development (Fgf4, Fgf17) or are not detectable during the initiation of genital outgrowth (Fgf9) (A. Seifert, M. Gredler, and M. J. Cohn, unpublished data). These findings highlight a key difference in the role of Fgf signaling between limbs and genitalia.
Wnt genes are good candidates for the proximodistal outgrowth signal, and Wnt2, Wnt3, Wnt4, Wnt5a, Wnt9b, and Wnt11 are expressed by E11.5, 1 day after initiation of budding (Lin et al., 2008). Conditional deletion of β-catenin in endodermal cells (using ShhGfpCre) results in failure to progress beyond the initiation of genital swellings (Lin et al., 2008). Removal of β-catenin activity results in diminished expression of a suite of genes, including Shh (described below), which led Lin et al to conclude that canonical Wnt signaling is essential for genital tubercle outgrowth (Lin et al., 2008). It should be noted that Wnt proteins are not the only factors that signal through β-catenin and, therefore, experimental modulation of β-catenin does not necessarily implicate Wnts in external genital development. Nonetheless, the role of Wnt/β-catenin is supported by two additional discoveries; (1) deletion of Lrp6, a co-receptor for canonical Wnt/β-catenin signaling, results in agenesis of external genitalia (Zhou et al., 2010), and (2) Dkk1, a negative regulator of canonical Wnt signaling, is expressed in the distal urethral epithelium and contains noncoding elements that can drive reporter gene expression in the genital tubercle (Lieven et al., 2010).
The role of noncanonical Wnt signaling in external genital development is less clear. Wnt5a was initially thought to be essential for genital tubercle outgrowth, however the penetrance of Wnt5a null allele is variable, and null mutants have been recovered with phenotypes ranging from rudimentary genital swellings to complete genital tubercles (Yamaguchi et al., 1999; Seifert et al., 2009b). Consistent with Wnt5a playing a role is the discovery that inactivation of Ror2, a Wnt5a receptor that mediates noncanonical Wnt signaling, results in underdevelopment of the external genitalia (Schwabe et al., 2004). Wnt11, which encodes another noncanonical Wnt signal, is expressed at the appropriate stages in external genital development to play a role in outgrowth, but its function is unknown (Lin et al., 2008).
Dynamic Role of Sonic Hedgehog in Outgrowth and Patterning
Before the onset of genital outgrowth, Shh is expressed in the cloacal epithelium, which gives rise to the urethral epithelium, and expression persists in urethral cells during the outgrowth period (Haraguchi et al., 2001; Perriton et al., 2002; Seifert et al., 2008). Although Shh mutant embryos do form the initial paired genital swellings, these buds arrest immediately after initiation and fail to give rise to a genital tubercle, which implicates Shh as having an essential role in the maintenance of genital outgrowth (Perriton et al., 2002). The Shh null phenotype can be partially rescued by over-expression of β-catenin in urethral epithelial cells, indicating that, although β-catenin signaling maintains expression of Shh in these cells, it can also promote limited outgrowth in the absence of Shh (Lin et al., 2009).
Conditional removal of Shh at different stages of genital tubercle development has provided a detailed picture of Shh function at daily intervals, from the initiation of budding through to stages of sexual differentiation (Lin et al., 2009; Miyagawa et al., 2009a; Seifert et al., 2009a). Shh regulates outgrowth by acting both as a proliferative cue and as a cell survival factor (Haraguchi et al., 2001; Perriton et al., 2002; Lin et al., 2009; Miyagawa et al., 2009a; Seifert et al., 2009a). Temporally controlled deletions showed that the extent of genital tubercle outgrowth is determined by the duration of Shh signaling, with earlier removals resulting in more severe truncations (Lin et al., 2009; Miyagawa et al., 2009a; Seifert et al., 2009a).
In addition to controlling proximodistal outgrowth, Shh is required for development of a closed urethral tube and for correct positioning of the urethral opening, as deletion of Shh up to E16.5 results in hypospadias, an ectopic opening of the urethra on the ventral side of the phallus (Lin et al., 2009; Miyagawa et al., 2009a; Seifert et al., 2009a). If Shh is removed before E13.5, mutants also develop a persistent cloaca, in which the embryonic cloaca fails to subdivide into anorectal and genitourinary sinuses. Removal of Shh after E13.5 does not affect division of the anorectal and genitourinary sinuses, but does result in severe hypospadias, which causes the genitalia to appear feminized (Seifert et al., 2009a).
Given that the genital tubercle consists of cells from endoderm, ectoderm, and mesoderm, which cells respond directly to Shh? Removal of Smoothened (Smo), which is required for activation of the hedgehog transduction pathway in response to hedgehog ligand, from either the genital mesenchyme or ectoderm disrupts genital development, whereas removal of Smo from the urethral epithelium has no effect on external genital morphology (Lin et al., 2009; Seifert et al., 2009a). These studies show that Shh does not act in an autocrine manner on Shh-expressing cells of the urethra, which is consistent with the absence of Patched1 (Ptch1) expression in these cells (Perriton et al., 2002; Lin et al., 2009; Seifert et al., 2009a). When Smo is removed from the mesenchyme, mice have severely underdeveloped genitalia and develop hypospadias (Lin et al., 2009). Removal of Smo from the ectoderm causes a loss of epithelial integrity ventral to the urethral plate, which results in an ectopic opening of the urethra along the ventral margin of the phallus (Seifert et al., 2009a). Thus, Shh signals directly to ectoderm and mesenchyme of the genital tubercle, and activation of the hedgehog pathway in both tissues is required for normal development of the external genitalia.
Whereas the role of Shh in genital ectoderm is to maintain structural integrity of the surface epithelium overlying the urethra, its major role in the mesenchyme is to inhibit apoptosis and to stimulate cell proliferation (Fig. 3). Shh promotes mesenchymal proliferation and tubercle outgrowth by controlling the duration of the cell cycle in the genital tubercle mesenchymal cells (Seifert et al., 2010). Deletion of Shh causes the mesenchymal cell cycle to slow to almost half-speed, which leads to grossly underdeveloped genitalia. Specifically, in Shh conditional mutants, mesenchymal cells spend an extended period in G1-phase before progressing to S-phase, which causes total cell cycle time to slow from 8.5 to 14.4 hr (Seifert et al., 2010). Shh directly and indirectly regulates several genes that govern cell cycle kinetics (Seifert et al., 2010) as well as pattern formation (Lin et al., 2009; Miyagawa et al., 2009a; Seifert et al., 2009a). The dual role of Shh—specification of early pattern and subsequent elaboration of pattern by growth regulation—appears to be common to limb and genital development (Towers et al., 2008; Zhu et al., 2008; Seifert et al., 2010). Given that integration of pattern formation and growth must occur for normal development of the limb and genital tubercle, this role of Shh may be necessary for outgrowth in many types of appendages.
Figure 3. Roles of Sonic hedgehog in the genital tubercle. Sonic hedgehog (Shh) expression in urethral epithelial cells is shown in blue. Mesodermal and surface ectodermal cells that respond directly to Shh signaling are shown in orange. Shh signals directly to the adjacent mesenchyme to positively regulate cell cycle progression (1) and to inhibit apoptosis (2). Shh signals directly to the ventral ectoderm to maintain the structural integrity of the epithelium (3), which is essential for maintenance of a closed urethral tube.
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The upstream regulation of Shh expression has been well characterized in the limb (Lettice et al., 2003; Galli et al., 2010), but remains poorly understood in the genital tubercle. By contrast to the limb, in which Shh and Fgf8 maintain each other in a positive feedback loop, loss of Fgf8 in the genital tubercle does not affect Shh expression. Fgf8 expression in the distal urethra is reduced in β-catenin and Shh mutants, although the discovery that Fgf8 is not involved in genital development indicates that Fgf8 transcription is simply a readout, rather than a cause, of genital outgrowth (Seifert et al., 2009b). Further analysis of Wnt gene function in early genital development should cast additional light on the interactions between specific Wnts and Sonic hedgehog.