The continuously growing mouse incisor is an excellent model to analyze the mechanisms for stem cell lineage. We designed an organ culture method for the apical end of the incisor and analyzed the epithelial cell lineage by 5-bromo-2′-deoxyuridine and DiI labeling. Our results indicate that stem cells reside in the cervical loop epithelium consisting of a central core of stellate reticulum cells surrounded by a layer of basal epithelial cells, and that they give rise to transit-amplifying progeny differentiating into enamel forming ameloblasts. We identified slowly dividing cells among the Notch1-expressing stellate reticulum cells in specific locations near the basal epithelial cells expressing lunatic fringe, a secretory molecule modulating Notch signaling. It is known from tissue recombination studies that in the mouse incisor the mesenchyme regulates the continuous growth of epithelium. Expression of Fgf-3 and Fgf-10 were restricted to the mesenchyme underlying the basal epithelial cells and the transit-amplifying cells expressing their receptors Fgfr1b and Fgfr2b. When FGF-10 protein was applied with beads on the cultured cervical loop epithelium it stimulated cell proliferation as well as expression of lunatic fringe. We present a model in which FGF signaling from the mesenchyme regulates the Notch pathway in dental epithelial stem cells via stimulation of lunatic fringe expression and, thereby, has a central role in coupling the mitogenesis and fate decision of stem cells.

The formation of periodic patterns is fundamental in biology. Theoretical models describing these phenomena have been proposed for feather patterning; however, no molecular candidates have been identified. Here we show that the feather tract is initiated by a continuous stripe of Shh, Fgf-4, and Ptc expression in the epithelium, which then segregates into discrete feather primordia that are more strongly Shh and Fgf-4 positive. The primordia also become Bmp-2 and Bmp-4 positive. Bead-mediated delivery of BMPs inhibits local feather formation in contrast with the activators, SHH and FGF-4, which induce feather formation. Both FGF-4 and SHH induce local expression of Bmp-4, while BMP-4 suppresses local expression of both. FGF-4 also induces Shh. Based on these findings, we propose a model that involves (1) homogeneously distributed global activators that define the field, (2) a position-dependent activator of competence that propagates across the field, and (3) local activators and inhibitors triggered in sites of individual primordia that act in a reaction-diffusion mechanism. A computer simulation model for feather pattern formation is also presented.

Primate molar shapes reflect developmental and ecological processes. Development may constrain as well as facilitate evolution of new tooth shapes, affecting how reliable dental characters are in phylogenetic studies. Much of the genetic machinery of development uses the same genes among different organs, including teeth, limbs, and feathers. Furthermore, within a tooth, the development of individual cusps repeatedly uses the same set of developmental genes, forming a "developmental module." The repeated activation of the developmental module can explain the cumulative variation in later-developing cusps. Therefore short, later-developing cusps may be evolvable but also more homoplastic. This patterning cascade mode of cusp development can be used to explain the variational properties of dental characters and character states related to cusp initiation. The developmental basis and variational properties of crown termination, cusp shape, and cusp configuration characters are currently less well understood. It is unlikely that there is a simple "gene to phenotype" map for dental characters. Rather, the whole cusp pattern is a product of a dynamic developmental program manifested in the activation of the developmental modules.

The Hippo signaling pathway regulates organ size, tissue regeneration, and stem cell self-renewal. The two key downstream transcription coactivators in this pathway, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), mediate the major gene regulation and biological functions of the Hippo pathway. The biological functions of YAP and TAZ in many tissues are known; however, their roles in skin wound healing remain unclear. To analyze whether YAP and/or TAZ are required for cutaneous wound healing, we performed small interfering RNA (siRNA)-mediated knockdown of YAP/TAZ in full-thickness skin wounds. YAP is strongly expressed in the nucleus and cytoplasm in the epidermis and hair follicle. Interestingly, YAP is expressed in the nucleus in the dermis at 2 and 7 days after wounding. TAZ normally localizes to the cytoplasm in the dermis but is distributed in both the nucleus and cytoplasm at 1 day after wounding. The knockdown of YAP and TAZ markedly delayed the rate of wound closure and reduced the transforming growth factor-β1 (TGF-β1) expression in the wound. YAP and TAZ also modulate the expression of TGF-β1 signaling pathway components such as Smad-2, p21, and Smad-7. These results suggest that YAP and TAZ localization to the nucleus is required for skin wound healing.

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.

Tabby is a mouse mutant characterized by deficient development of the ectodermal organs: teeth, hair, and a subset of glands. Ectodysplasin, the protein encoded by the Tabby gene, was recently identified as a novel TNF-like transmembrane protein but little is known about its function. We have examined the Tabby tooth phenotype in detail by analysis of the adult and embryonic teeth. Tabby first molars had an obvious defect in cusp patterning as the number of cusps was reduced and the buccal and lingual cusps were joined. The disturbance in development was first visible morphologically in the bud stage molar. The primary enamel knot in a cap stage Tabby tooth expressed all enamel knot markers analyzed but was smaller than wild type and the first pair of developing secondary enamel knots was fused. We propose that the Tabby tooth phenotype is due to growth retardation during early stages of development which leads to reduced signaling from the primary enamel knot, followed by deficient growth of the dental epithelium and lack of formation of the last developing secondary enamel knots. The ectodysplasin transcripts were expressed in the outer enamel epithelium and dental lamina. When cultured in vitro Tabby bud/cap stage molars formed fewer cusps than wild-type controls. This phenotype was not rescued by exogenously added EGF despite the previously proposed link between Tabby and EGF. Instead FGF-10 partially restored morphogenesis and stimulated the development of additional tooth cusps in cultured Tabby molars.

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We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2.