Dosage-dependent hedgehog signals integrated with Wnt/β-catenin signaling regulate external genitalia formation as an appendicular program

Abstract

Embryonic appendicular structures, such as the limb buds and the developing external genitalia, are suitable models with which to analyze the reciprocal interactions of growth factors in the regulation of outgrowth. Although several studies have evaluated the individual functions of different growth factors in appendicular growth, the coordinated function and integration of input from multiple signaling cascades is poorly understood. We demonstrate that a novel signaling cascade governs formation of the embryonic external genitalia [genital tubercle (GT)]. We show that the dosage of Shh signal is tightly associated with subsequent levels of Wnt/-catenin activity and the extent of external genitalia outgrowth. In Shh-null mouse embryos, both expression of Wnt ligands and Wnt/-catenin signaling activity are downregulated. -catenin gain-of-function mutation rescues defective GT outgrowth and Fgf8 expression in Shh-null embryos. These data indicate that Wnt/-catenin signaling in the distal urethral epithelium acts downstream of Shh signaling during GT outgrowth. The current data also suggest that Wnt/-catenin regulates Fgf8 expression via Lef/Tcf binding sites in a 3Ј conserved enhancer. Fgf8 induces phosphorylation of Erk1/2 and cell proliferation in the GT mesenchyme in vitro, yet Fgf4/8 compound-mutant phenotypes indicate dispensable functions of Fgf4/8 and the possibility of redundancy among multiple Fgfs in GT development. Our results provide new insights into the integration of growth factor signaling in the appendicular developmental programs that regulate external genitalia development.

Timing of androgen receptor disruption and estrogen exposure underlies a spectrum of congenital penile anomalies

“…By contrast, conditional deletion of AR in urethral epithelial cells had no affect on genital development or sexual differentiation, suggesting that cell death at the ventral ectoderm-endoderm junction may be mediated via the adjacent mesenchyme rather than by a cell autonomous response of urethral endodermal or ventral ectodermal cells to AR activity. Our hypothesis that AR acts primarily on genital mesenchyme and that a relay mechanism mediates its effect on adjacent epithelia is supported by evidence that androgen signaling regulates expression of genes in the Fgf, Bmp, and Hh pathways, which are known to control apoptosis during early development of the genital tubercle (35,37,39,40,43).…”

“…Based on the discovery that androgen and estrogen control sexual differentiation of external genitalia by regulating urethral epithelial apoptosis in the embryo and mesenchymal cell proliferation in the neonate and the observation that male and female genitalia show differences in AR and ERα activity at these two stages, we tested the hypothesis that the gene network controlling early genital development may become differentially regulated in males and females during sexual differentiation. Given that the Hedgehog, bone morphogenetic protein (Bmp), Wnt, and fibroblast growth factor (Fgf) signaling pathways play key roles in genital development and that mutations of genes in these pathways result in malformations of external genitalia (13,(35)(36)(37)(38)(39)(40)(41), we tested whether AR or ERα signaling regulates the expression levels of genes in these pathways. Quantification of the transcript levels of 88 genes in males treated with flutamide and in control males at E15.5 identified 22 genes with significant responses to flutamide (Fig.…”

Sexually dimorphic expression of Mafb regulates masculinization of the embryonic urethral formation

“…Another characteristic of the masculinization of external genitalia is the difference in the GT organ size. Regulation of cell proliferation by growth factors has been suggested as an essential process for the GT development (30,(34)(35)(36). Genedriven GT outgrowth regulated by several growth factors has been proposed as essential for androgen-independent early embryonic GT growth.…”

FGF4 and FGF8 comprise the wavefront activity that controls somitogenesis

“…It is now clear that FGF4 and FGF8 are the principal FGFs required for both axis extension and limb bud outgrowth (29,37). Both genes are also expressed in the genital tubercle (GT), and whereas GT-specific inactivation of Fgf4 and Fgf8 shows that they are dispensable for GT outgrowth, this may be due to increased activity of other ligands (38). Furthermore, in all three systems, FGF activity is regulated by WNT signaling (38,39), and the coordinate WNT/FGF signaling system may have evolved in early chordates to link the somite clock to body axis extension (40).…”

“…Both genes are also expressed in the genital tubercle (GT), and whereas GT-specific inactivation of Fgf4 and Fgf8 shows that they are dispensable for GT outgrowth, this may be due to increased activity of other ligands (38). Furthermore, in all three systems, FGF activity is regulated by WNT signaling (38,39), and the coordinate WNT/FGF signaling system may have evolved in early chordates to link the somite clock to body axis extension (40). These data support speculations that programs controlling axis and tail extension have been co-opted over the course of evolution to control the outgrowth of multiple appendages (41,42).…”