External genitalia are body appendages specialized for internal fertilization. Their development can be divided into two phases, an early androgen-independent phase and a late androgen-dependent sexual differentiation phase. In the early phase, the embryonic anlage of external genitalia, the genital tubercle (GT), is morphologically identical in both sexes. Although congenital external genitalia malformations represent the second most common birth defect in humans, the genetic pathways governing early external genitalia development and urethra formation are poorly understood. Proper development of the GT requires coordinated outgrowth of the mesodermally derived mesenchyme and extension of the endodermal urethra within an ectodermal epithelial capsule. Here, we demonstrate that β-catenin plays indispensable and distinct roles in each of the aforementioned three tissue layers in early androgen-independent GT development. WNT-β-catenin signaling is required in the endodermal urethra to activate and maintain Fgf8 expression and direct GT outgrowth, as well as to maintain homeostasis of the urethra. Moreover, β-catenin is required in the mesenchyme to promote cell proliferation. By contrast, β-catenin is required in the ectoderm to maintain tissue integrity, possibly through cell-cell adhesion during GT outgrowth. The fact that both endodermal and ectodermal β-catenin knockout animals develop severe hypospadias in both sexes raises the possibility that the deregulation of any of these functions can contribute to the etiology of congenital external genital defects in humans.

Genital tubercle (GT) initiation and outgrowth involve coordinated morphogenesis of surface ectoderm, cloacal mesoderm and hindgut endoderm. GT development appears to mirror that of the limb. Although Shh is essential for the development of both appendages, its role in GT development is much less clear than in the limb. Here, by removing Shh at different stages during GT development in mice, we demonstrate a continuous requirement for Shh in GT initiation and subsequent androgen-independent GT growth. Moreover, we investigated the Hh responsiveness of different tissue layers by removing or activating its signal transducer Smo with tissue-specific Cre lines, and established GT mesenchyme as the primary target tissue of Shh signaling. Lastly, we showed that Shh is required for the maintenance of the GT signaling center distal urethral epithelium (dUE). By restoring Wnt-Fgf8 signaling in Shh-/- cloacal endoderm genetically, we revealed that Shh relays its signal partly through the dUE, but regulates Hoxa13 and Hoxd13 expression independently of dUE signaling. Altogether, we propose that Shh plays a central role in GT development by simultaneously regulating patterning of the cloacal field and supporting an outgrowth signal.

The Cullin-RING ubiquitin-ligase CRL4 controls cell cycle, DNA damage checkpoint response and ensures genomic integrity. Inactivation of the Cul4 component of the CRL4 E3 ligase complex in Caenorhabditis elegans by RNA interference results in massive mitotic DNA re-replication in the blast cells, largely due to failed degradation of the DNA licensing protein, CDT-1, and premature spermatogenesis. Here we show that inactivation of Cul4a by gene-targeting in mice only affected male but not female fertility. This male infertility phenotype resulted from a combination of decreased spermatozoa number, reduced sperm motility and defective acrosome formation. Agenesis of the mutant germ cells was accompanied by increased cell death in pachytene/diplotene cells with markedly elevated levels of phospho-p53 and CDT-1. Despite apparent normal assembly of synaptonemal complexes and DNA double strand break repair, dissociation of MLH1, a component of the late recombination nodule, was delayed in Cul4a−/− diplotene spermatocytes, which potentially led to subsequent disruptions in meiosis II and spermiogenesis. Together, our study revealed an indispensable role for Cul4a during male germ cell meiosis.

The acquisition of the external genitalia allowed mammals to cope with terrestrial-specific reproductive needs for internal fertilization, and thus it represents one of the most fundamental steps in evolution towards a life on land. How genitalia evolved remains obscure, and the key to understanding this process may lie in the developmental genetics that underpins the early establishment of the genital primordium, the genital tubercle (GT). Development of the GT is similar to that of the limb, which requires precise regulation from a distal signaling epithelium. However, whether outgrowth of the GT and limbs is mediated by common instructive signals remains unknown. In this study, we used comprehensive genetic approaches to interrogate the signaling cascade involved in GT formation in comparison with limb formation. We demonstrate that the FGF ligand responsible for GT development is FGF8 expressed in the cloacal endoderm. We further showed that forced Fgf8 expression can rescue limb and GT reduction in embryos deficient in WNT signaling activity. Our studies show that the regulation of Fgf8 by the canonical WNT signaling pathway is mediated in part by the transcription factor SP8. Sp8 mutants elicit appendage defects mirroring WNT and FGF mutants, and abolishing Sp8 attenuates ectopic appendage development caused by a gain-of-function β-catenin mutation. These observations indicate that a conserved WNT-SP8-FGF8 genetic cassette is employed by both appendages for promoting outgrowth, and suggest a deep homology shared by the limb and external genitalia.

Proper patterning and growth of oral structures including teeth, tongue, and palate rely on epithelial-mesenchymal interactions involving coordinated regulation of signal transduction. Understanding molecular mechanisms underpinning oral-facial development will provide novel insights into the etiology of common congenital defects such as cleft palate. In this study, we report that ablating Wnt signaling in the oral epithelium blocks the formation of palatal rugae, which are a set of specialized ectodermal appendages serving as Shh signaling centers during development and niches for sensory cells and possibly neural crest related stem cells in adults. Lack of rugae is also associated with retarded anteroposterior extension of the hard palate and precocious mid-line fusion. These data implicate an obligatory role for canonical Wnt signaling in rugae development. Based on this complex phenotype, we propose that the sequential addition of rugae and its morphogen Shh, is intrinsically coupled to the elongation of the hard palate, and is critical for modulating the growth orientation of palatal shelves. In addition, we observe a unique cleft palate phenotype at the anterior end of the secondary palate, which is likely caused by the severely underdeveloped primary palate in these mutants. Last but not least, we also discover that both Wnt and Shh signalings are essential for tongue development. We provide genetic evidence that disruption of either signaling pathway results in severe microglossia. Altogether, we demonstrate a dynamic role for Wnt-β-Catenin signaling in the development of the oral apparatus.

SUMMARYDevelopment of the metanephric kidney depends on precise control of branching of the ureteric bud. Branching events represent terminal bifurcations that are thought to depend on unique patterns of gene expression in the tip compared with the stalk and are influenced by mesenchymal signals. The metanephric mesenchyme-derived signals that control gene expression at the ureteric bud tip are not well understood. In mouse Sall1 mutants, the ureteric bud grows out and invades the metanephric mesenchyme, but it fails to initiate branching despite tip-specific expression of Ret and Wnt11. The stalk-specific marker Wnt9b and the -catenin downstream target Axin2 are ectopically expressed in the mutant ureteric bud tips, suggesting that upregulated canonical Wnt signaling disrupts ureter branching in this mutant. In support of this hypothesis, ureter arrest is rescued by lowering -catenin levels in the Sall1 mutant and is phenocopied by ectopic expression of a stabilized -catenin in the ureteric bud. Furthermore, transgenic overexpression of Wnt9b in the ureteric bud causes reduced branching in multiple founder lines. These studies indicate that Sall1-dependent signals from the metanephric mesenchyme are required to modulate ureteric bud tip Wnt patterning in order to initiate branching.

In utero exposure to diethylstilbestrol (DES) leads to patterning defects in the female reproductive tract (FRT) and a propensity to the development of vaginal adenocarcinomas in humans. In the mouse, DES treatment similarly induces a plethora of FRT developmental defects, including stratification of uterine epithelium and presence of glandular tissue in cervix and vagina. Uterine abnormalities are associated with repression of the homeobox gene Msx2, and DES leads to an altered uterine response in Msx2 mutants including a dilated uterine lumen. Here we investigate the role of Msx2 in normal vaginal development and in FRT response to DES. During vaginal development, Msx2 is required for Tgfbeta2 and Tgfbeta3 expression and for proper vaginal epithelial differentiation. Moreover, Msx2 is involved in caudal Wolffian duct regression by promoting apoptosis. Consistently, neonatal DES exposure represses Msx2 expression in the Wolffian duct epithelium and inhibits its apoptosis and subsequent regression. Intriguingly, although DES treatment also represses Msx2 expression in the vaginal epithelium, a much more severe DES-induced vaginal phenotype was observed in Msx2 mutant mice, including a complete failure of Müllerian vaginal epithelial stratification and a severely dilated vaginal lumen, accompanied by loss of p63 and water channel protein expression. These results demonstrate a critical role for Msx2 in counteracting the effect of DES on FRT patterning and suggest that the response to DES may be highly variable depending on the genotype of an individual.

The incidence for bladder urothelial carcinoma (UC), a common malignancy of the urinary tract, is about three times higher in men than in women. Although this gender difference has been primarily attributed to differential exposures, it is likely that underlying biological causes contribute to the gender inequality. In this study, we report a transgenic mouse bladder tumor model upon induction of constitutively activated β-catenin signaling in the adult urothelium. We showed that the histopathology of the tumors observed in our model closely resembled that of the human low grade urothelial carcinoma. Additionally, we provided evidence supporting the KRT5-positive;KRT7-negative basal cells as the putative cells-of-origin for β-catenin-induced luminal tumor. Intriguingly, the tumorigenesis in this model demonstrated a marked difference between opposite sexes; forty percent of males developed macroscopically detectable luminal tumors in twelve weeks, whereas only three percent of females developed tumors. We investigated the mechanisms underlying this sexual dimorphism in pathogenesis and demonstrated that nuclear translocation of the androgen receptor (AR) in the urothelial cells is a critical mechanism contributing to tumor development in male mice. Finally, we performed global gene profiling experiments and defined the molecular signature for the β-catenin-induced tumorigenesis in males. Altogether, we have established a model for investigating sexual dimorphism in UC development, and implicated synergy between β-catenin signaling and androgen/AR signaling in carcinogenesis of the basal urothelial cells.