Osteoclasts form an acidic compartment at their attachment site in which bone demineralization and matrix degradation occur. Although both the cysteine proteinases and neutral collagenases participate in bone resorption, their roles have remained unclear. Here we show that interstitial collagenase has an essential role in initiating bone resorption, distinct from that of the cysteine proteinases. Treatment of osteoclasts with cysteine proteinase inhibitors did not affect the number of resorption lacunae ("pits") formed on the surface of dentine slices, but it generated abnormal pits that were demineralized but filled with undegraded matrix. Treatment with metalloproteinase inhibitors did not alter the qualitative features of lacunae, but it greatly reduced the number of pits and surface area resorbed. Treatment of bone cells with an inhibitory anti-rat interstitial collagenase antiserum reduced bone resorption markedly. In the presence of collagenase inhibitors, resorption was restored by pretreatment of dentine slices with rat interstitial collagenase or by precoating the dentine slices with collagenase-derived gelatin peptides or heatgelatinized collagen. Immunostaining revealed that interstitial collagenase is produced at high levels by stromal cells and osteoblasts adjacent to osteoclasts. These results indicate that interstitial collagenase can function as a "coupling factor," allowing osteoblasts to initiate bone resorption by generating collagen fragments that activate osteoclasts.Normal bone turnover is highly regulated. Osteoclasts, the cells that degrade bone, require activation to trigger their boneresorptive capacity. Once activated, osteoclasts secrete both protons and proteinases at their attachment site, resulting in dissolution of bone mineral and degradation of the matrix (1). Osteoclasts produce several cysteine proteinases (2-4), enzymes with acidic pH optima, of which cathepsin K appears to be essential for normal bone resorption (5, 6). Studies indicate that the major function of secreted cysteine proteinases is matrix degradation (3, 7).The role of neutral metalloproteinases, a second class of proteinase produced in bone tissue (3,8), is less clear. Members of the metalloproteinase family have neutral pH optima, are secreted as proenzymes, and contain a zinc atom at the active site (9, 10). Although some prior studies suggested that neutral metalloproteinases contribute to osteoclast matrix degradation (11), recent evidence indicates that osteoclasts do not produce collagenase (12). Collagenase is produced by cells of osteoblastic lineage and may be required for resorption of intact bone tissue (13)(14)(15)(16). Observations that isolated osteoclasts that had no detectable collagenase activity were able to resorb bone prompted the suggestion that collagenase promotes resorption by removing unmineralized matrix from the bone surface, facilitating osteoclast attachment (8,17,18).In this study we have examined the qualitative and quantitative roles of acid cysteine proteinases and interstitial...

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.