SUMMARYEach vertebrate species displays specific tooth patterns in each quadrant of the jaw: the mouse has one incisor and three molars, which develop at precise locations and at different times. The reason why multiple teeth form in the jaw of vertebrates and the way in which they develop separately from each other have been extensively studied, but the genetic mechanism governing the spatial patterning of teeth still remains to be elucidated. Sonic hedgehog (Shh) is one of the key signaling molecules involved in the spatial patterning of teeth and other ectodermal organs such as hair, vibrissae and feathers. Sostdc1, a secreted inhibitor of the Wnt and Bmp pathways, also regulates the spatial patterning of teeth and hair. Here, by utilizing maternal transfer of 5E1 (an anti-Shh antibody) to mouse embryos through the placenta, we show that Sostdc1 is downstream of Shh signaling and suggest a Wnt-Shh-Sostdc1 negative feedback loop as a pivotal mechanism controlling the spatial patterning of teeth. Furthermore, we propose a new reaction-diffusion model in which Wnt, Shh and Sostdc1 act as the activator, mediator and inhibitor, respectively, and confirm that such interactions can generate the tooth pattern of a wild-type mouse and can explain the various tooth patterns produced experimentally.

Various cellular and molecular events underlie the elevation and fusion of the developing palate that occurs during embryonic development. This includes convergent extension, where the medial edge epithelium is intercalated into the midline epithelial seam. We examined the expression patterns of Wnt11 and Fgfr1b - which are believed to be key factors in convergent extension - in mouse palate development. Wnt-11 overexpression and beads soaked in SU5402 (an Fgfr1 inhibitor) were employed in in vitro organ cultures. The results suggested that interactions between Wnt11 and Fgfr1b are important in modulating cellular events such as cell proliferation for growth and apoptosis for fusion. Moreover, the Wnt11 siRNA results showed that Wnt11-induced apoptosis was necessary for palatal fusion. In summary, Fgfr1b induces cell proliferation in the developing palate mesenchyme so that the palate grows and contacts each palatal shelf, with negative feedback of Fgfs triggered by excessive cell proliferation then inhibiting the expression of Fgfr1b and activating the expression of Wnt11 to fuse each palate by activating apoptosis.

Inhibitor of differentiation 1 (ID1) is highly expressed in glioblastoma stem cells (GSCs). However, the regulatory mechanism responsible for its role in GSCs is poorly understood. Here, we report that ID1 activates GSC proliferation, self-renewal, and tumorigenicity by suppressing CULLIN3 ubiquitin ligase. ID1 induces cell proliferation through increase of CYCLIN E, a target molecule of CULLIN3. ID1 overexpression or CULLIN3 knockdown confers GSC features and tumorigenicity to murine Ink4a/Arf-deficient astrocytes. Proteomics analysis revealed that CULLIN3 interacts with GLI2 and DVL2 and induces their degradation via ubiquitination. Consistent with ID1 knockdown or CULLIN3 overexpression in human GSCs, pharmacologically combined control of GLI2 and β-CATENIN effectively diminishes GSC properties. A ID1-high/CULLIN3-low expression signature correlates with a poor patient prognosis, supporting the clinical relevance of this signaling axis. Taken together, a loss of CULLIN3 represents a common signaling node for controlling the activity of intracellular WNT and SHH signaling pathways mediated by ID1.

Various cellular and molecular events are involved in palatogenesis, including apoptosis, epithelial-mesenchymal transition (EMT), cell proliferation, and cell migration. Smad2 and Snail, which are well-known key mediators of the transforming growth factor beta (Tgf-β) pathway, play a crucial role in the regulation of palate development. Regulatory effects of microRNA 200b (miR-200b) on Smad2 and Snail in palatogenesis have not yet been elucidated. The aim of this study is to determine the relationship between palate development regulators miR-200b and Tgf-β-mediated genes. Expression of miR-200b, E-cadherin, Smad2, and Snail was detected in the mesenchyme of the mouse palate, while miR-200b was expressed in the medial edge epithelium (MEE) and palatal mesenchyme. After the contact of palatal shelves, miR-200b was no longer expressed in the mesenchyme around the fusion region. The binding activity of miR-200b to both Smad2 and Snail was examined using a luciferase assay. MiR-200b directly targeted Smad2 and Snail at both cellular and molecular levels. The function of miR-200b was determined by overexpression via a lentiviral vector in the palatal shelves. Ectopic expression of miR-200b resulted in suppression of these Tgf-β-mediated regulators and changes of apoptosis and cell proliferation in the palatal fusion region. These results suggest that miR-200b plays a crucial role in regulating the Smad2, Snail, and in apoptosis during palatogenesis by acting as a direct non-coding, influencing factor. Furthermore, the molecular interactions between miR-200b and Tgf-β signaling are important for proper palatogenesis and especially for palate fusion. Elucidating the mechanism of palatogenesis may aid the design of effective gene-based therapies for the treatment of congenital cleft palate.

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Teeth, an excellent model for studying organogenesis, develop from a series of epithelial-mesenchymal interactions that are mediated by a complex molecular network. Bcor (BCL-6 interacting corepressor) has recently been discovered, but little is known about its function in tooth development. Mutations in BCOR affect humans with oculofaciocardiodental syndrome, which is an X-linked dominant disorder with presumed male lethality and which comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. In this study, the Bcor expression pattern has been intensively investigated during mouse molar development. Bcor is expressed in both dental epithelium and the mesenchyme at E11.5. To understand the function of Bcor, knockdown of Bcor has been examined by using lentivirus-mediated RNA interference. Silencing of Bcor expression in dental mesenchymal cells at E14.5 causes dentinogenesis defects and retardation of tooth root development. Thus, our results suggest that Bcor expressed in the mesenchyme plays crucial roles during early tooth development. The function of Bcor expressed in the epithelium remains to be elucidated.

Although tridermic species have two junctional regions of ectoderm and endoderm between their epidermis and digestive tract, we actually know little about these particular boundaries. Cytokeratins are the major intermediate filaments of epithelial cells and show a high degree of tissue specificity. Therefore, to characterize the epithelial cells in the junctional region of ectoderm and endoderm, we immunohistochemically examined the localization of cytokeratins 5, 7/17, 14, 18, Sox17, and alpha-fetoprotein (AFP) in the oropharyngeal and anorectal regions during the mouse gastrulation process. At embryonic day (E) 9.5, cytokeratins 5, 7/17, 14, and 18 were detected in all epithelial cells of the oropharyngeal region. At E12.5, cytokeratin 5-positive cells were not observed in the middle area of the oral cavity; however, the immunoreactivity was strong in the anterior and posterior areas. The immunoreaction of cytokeratins 18 was seen only in the middle and posterior areas of the oral mucosa. Cytokeratins 7/17 and 14 were localized in all areas of the oropharyngeal region. Sox17 and AFP, which are endodermal markers, were detected in the middle and posterior areas of the oral mucosa, but not in the anterior area. Moreover, this same localization pattern of cytokeratins also existed in the anorectal region of the E12.5 embryo, suggesting that the localization of cytokeratins and endodermal markers might give an implication for the boundary between ectoderm and endoderm. These results also suggest that these cytokeratins are useful molecules for monitoring the epithelial cell differentiation in the junctional region of the germ layers.