Delineating a conserved genetic cassette promoting outgrowth of body appendages.

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 ß-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.

Neonatal diethylstilbestrol exposure alters the metabolic profile of uterine epithelial cells.

Developmental exposure to diethylstilbestrol (DES) causes reproductive tract malformations, affects fertility and increases the risk of clear cell carcinoma of the vagina and cervix in humans. Previous studies on a well-established mouse DES model demonstrated that it recapitulates many features of the human syndrome, yet the underlying molecular mechanism is far from clear. Using the neonatal DES mouse model, the present study uses global transcript profiling to systematically explore early gene expression changes in individual epithelial and mesenchymal compartments of the neonatal uterus. Over 900 genes show differential expression upon DES treatment in either one or both tissue layers. Interestingly, multiple components of peroxisome proliferator-activated receptor-γ (PPARγ)-mediated adipogenesis and lipid metabolism, including PPARγ itself, are targets of DES in the neonatal uterus. Transmission electron microscopy and Oil-Red O staining further demonstrate a dramatic increase in lipid deposition in uterine epithelial cells upon DES exposure. Neonatal DES exposure also perturbs glucose homeostasis in the uterine epithelium. Some of these neonatal DES-induced metabolic changes appear to last into adulthood, suggesting a permanent effect of DES on energy metabolism in uterine epithelial cells. This study extends the list of biological processes that can be regulated by estrogen or DES, and provides a novel perspective for endocrine disruptor-induced reproductive abnormalities.

The inductive role of Wnt-ß-Catenin signaling in the formation of oral apparatus.

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-ß-Catenin signaling in the development of the oral apparatus.

Temporal and spatial dissection of Shh signaling in genital tubercle development.

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.