Autocrine and paracrine Shh signaling are necessary for tooth morphogenesis, but not tooth replacement in snakes and lizards (Squamata)

Here we study the role of Shh signaling in tooth morphogenesis and successional tooth initiation in snakes and lizards (Squamata). By characterizing the expression of Shh pathway receptor Ptc1 in the developing dentitions of three species (Eublepharis macularius, Python regius, and Pogona vitticeps) and by performing gain- and loss-of-function experiments, we demonstrate that Shh signaling is active in the squamate tooth bud and is required for its normal morphogenesis. Shh apparently mediates tooth morphogenesis by separate paracrine- and autocrine-mediated functions. According to this model, paracrine Shh signaling induces cell proliferation in the cervical loop, outer enamel epithelium, and dental papilla. Autocrine signaling within the stellate reticulum instead appears to regulate cell survival. By treating squamate dental explants with Hh antagonist cyclopamine, we induced tooth phenotypes that closely resemble the morphological and differentiation defects of vestigial, first-generation teeth in the bearded dragon P. vitticeps. Our finding that these vestigial teeth are deficient in epithelial Shh signaling further corroborates that Shh is needed for the normal development of teeth in snakes and lizards. Finally, in this study, we definitively refute a role for Shh signaling in successional dental lamina formation and conclude that other pathways regulate tooth replacement in squamates.     

A new function of BMP4: dual role for BMP4 in regulation of Sonic hedgehog expression in the mouse tooth germ

The murine tooth development is governed by sequential and reciprocal epithelial-mesenchymal interactions. Multiple signaling molecules are expressed in the developing tooth germ and interact each other to mediate the inductive tissue interactions. Among them are Sonic hedgehog (SHH), Bone Morphogenetic Protein-2 (BMP2) and Bone Morphogenetic Protein-4 (BMP4). We have investigated the interactions between these signaling molecules during early tooth development. We found that the expression of Shh and Bmp2 is downregulated at E12.5 and E13.5 in the dental epithelium of the Msx1 mutant tooth germ where Bmp4 expression is significantly reduced in the dental mesenchyme. Inhibition of BMP4 activity by noggin resulted in repression of Shh and Bmp2 in wild-type dental epithelium. When implanted into the dental mesenchyme of Msx1 mutants, beads soaked with BMP4 protein were able to restore the expression of both Shh and Bmp2 in the Msx1 mutant epithelium. These results demonstrated that mesenchymal BMP4 represents one component of the signal acting on the epithelium to maintain Shh and Bmp2 expression. In contrast, BMP4-soaked beads repressed Shh and Bmp2 expression in the wild-type dental epithelium. TUNEL assay indicated that this suppression of gene expression by exogenous BMP4 was not the result of an increase in programmed cell death in the tooth germ. Ectopic expression of human Bmp4 to the dental mesenchyme driven by the mouse Msx1 promoter restored Shh expression in the Msx1 mutant dental epithelium but repressed Shh in the wild-type tooth germ in vivo. We further demonstrated that this regulation of Shh expression by BMP4 is conserved in the mouse developing limb bud. In addition, Shh expression was unaffected in the developing limb buds of the transgenic mice in which a constitutively active Bmpr-IB is ectopically expressed in the forelimb posterior mesenchyme and throughout the hindlimb mesenchyme, suggesting that the repression of Shh expression by BMP4 may not be mediated by BMP receptor-IB. These results provide evidence for a new function of BMP4. BMP4 can act upstream to Shh by regulating Shh expression in mouse developing tooth germ and limb bud. Taken together, our data provide insight into a new regulatory mechanism for Shh expression, and suggest that this BMP4-mediated pathway in Shh regulation may have a general implication in vertebrate organogenesis.     

Shh signaling within the dental epithelium is necessary for cell proliferation,growth and polarization

Sonic hedgehog (Shh), a member of the mammalian Hedgehog (Hh) family, plays a key role during embryogenesis and organogenesis. Tooth development, odontogenesis, is governed by sequential and reciprocal epithelial-mesenchymal interactions. Genetic removal of Shh activity from the dental epithelium, the sole source of Shh during tooth development, alters tooth growth and cytological organization within both the dental epithelium and mesenchyme of the tooth. In this model it is not clear which aspects of the phenotype are the result of the direct action of Shh on a target tissue and which are indirect effects due to deficiencies in reciprocal signalings between the epithelial and mesenchymal components. To distinguish between these two alternatives and extend our understanding of Shh's actions in odontogenesis, we have used the Cre-loxP system to remove Smoothened (Smo) activity in the dental epithelium. Smo, a seven-pass membrane protein is essential for the transduction of all Hh signals. Hence, removal of Smo activity from the dental epithelium should block Shh signaling within dental epithelial derivatives while preserving normal mesenchymal signaling. Here we show that Shh-dependent interactions occur within the dental epithelium itself. The dental mesenchyme develops normally up until birth. In contrast, dental epithelial derivatives show altered proliferation, growth, differentiation and polarization. Our approach uncovers roles for Shh in controlling epithelial cell size, organelle development and polarization. Furthermore, we provide evidence that Shh signaling between ameloblasts and the overlying stratum intermedium may involve subcellular localization of Patched 2 and Gli1 mRNAs, both of which are targets of Shh signaling in these cells.     

Sonic hedgehog regulates growth and morphogenesis of the tooth

During mammalian tooth development, the oral ectoderm and mesenchyme coordinate their growth and differentiation to give rise to organs with precise shapes, sizes and functions. The initial ingrowth of the dental epithelium and its associated dental mesenchyme gives rise to the tooth bud. Next, the epithelial component folds to give the tooth its shape. Coincident with this process, adjacent epithelial and mesenchymal cells differentiate into enamel-secreting ameloblasts and dentin-secreting odontoblasts, respectively. Growth, morphogenesis and differentiation of the epithelium and mesenchyme are coordinated by secreted signaling proteins. Sonic hedgehog (Shh) encodes a signaling peptide which is present in the oral epithelium prior to invagination and in the tooth epithelium throughout its development. We have addressed the role of Shh in the developing tooth in mouse by using a conditional allele to remove Shh activity shortly after ingrowth of the dental epithelium. Reduction and then loss of Shh function results in a cap stage tooth rudiment in which the morphology is severely disrupted. The overall size of the tooth is reduced and both the lingual epithelial invagination and the dental cord are absent. However, the enamel knot, a putative organizer of crown formation, is present and expresses Fgf4, Wnt10b, Bmp2 and Lef1, as in the wild type. At birth, the size and the shape of the teeth are severely affected and the polarity and organization of the ameloblast and odontoblast layers is disrupted. However, both dentin- and enamel-specific markers are expressed and a large amount of tooth-specific extracellular matrix is produced. This observation was confirmed by grafting studies in which tooth rudiments were cultured for several days under kidney capsules. Under these conditions, both enamel and dentin were deposited even though the enamel and dentin layers remained disorganized. These studies demonstrate that Shh regulates growth and determines the shape of the tooth. However, Shh signaling is not essential for differentiation of ameloblasts or odontoblasts.     

Shh and ROCK1 modulate the dynamic epithelial morphogenesis in circumvallate papilla development

In rodents, a circumvallate papilla (CVP) develops with dynamic changes in epithelial morphogenesis during early tongue development. Molecular and cellular studies of CVP development revealed that there would be two different mechanisms in the apex and the trench wall forming regions with specific expression patterns of Wnt11 and Shh. Molecular interactions were examined using in vitro organ culture with over-expression of Shh, important signalling molecules and various inhibitors revealed that there are two significant different mechanisms in CVP formation by Wnt11 and Shh expressions. Wnt, a well known key molecule to initiate taste papillae, would govern Rho activation and cytoskeleton formation in the apex epithelium of CVP. In contrast, Shh regulates the cell proliferation to differentiate taste buds and to invaginate the epithelium for development of von Ebner's gland (VEG). Based on these results, we suggest that these different molecular signalling cascades of Wnt11 and Shh would play crucial roles in specific morphogenesis and pattern formation of CVP during early mouse embryo development.     

Tooth regeneration from newly established cell lines from a molar tooth germ epithelium

In order to investigate tooth development, several cell lines of the dental epithelium and ectomesenchyme have been established. However, no attempt has been reported to regenerate teeth with cell lines. Here, we have established several clonal cell lines of the dental epithelium from a p53-deficient fetal mouse. They expressed specific markers of the dental epithelium such as ameloblastin and amelogenin. A new method has been developed to bioengineer tooth germs with dental epithelial and mesenchymal cells. Reconstructed tooth germs with cell lines and fetal mesenchymal cells were implanted under kidney capsule. The germs regenerated teeth with well-calcified structures as seen in natural tooth. Germs without the cell lines developed bone. This is the first success to regenerate teeth with dental epithelial cell lines. They are useful models in vitro for investigation of mechanisms in morphogenesis and of cell lineage in differentiation, and for clinical application for tooth regeneration.     

Deletion of the Pitx1 genomic locus affects mandibular tooth morphogenesis and expression of the Barx1 and Tbx1 genes

Pitx1 is a bicoid-related homeodomain factor that exhibits preferential expression in the developing hindlimb, mandible, pituitary gland and teeth. Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of both hindlimb and mandible, suggesting a proliferative defect in these two structures. Here, we studied the expression and regulation of Pitx1 in both mandible and developing teeth and analyzed tooth morphology, cell proliferation, apoptosis and expression of Pitx2, Barx1 and Tbx1 in dental tissues of Pitx1−/− mouse embryos. Pitx1 expression is restricted to the epithelium of the growing tooth anlagen. Tissue recombination and bead implantation experiments demonstrated that bone morphogenetic protein-4 down-regulates Pitx1 expression in both mandibular mesenchyme and dental epithelium. Deletion of the Pitx1 locus results in micrognathia and abnormal morphology of the mandibular molars. Although Pitx2 expression in teeth of Pitx1−/− embryos is not altered, expression of Barx1 decreased in the mesenchyme of the mandibular molars. Furthermore, Pitx1 deletion results in suppression of Tbx1 expression in dental epithelium. Taken together, these results indicate that independent genetic pathways in mandibular and maxillary processes determine tooth development and morphology.     

Laminin alpha5 is required for dental epithelium growth and polarity and the development of tooth bud and shape

In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha5 (Lama5) in tooth development using laminin alpha5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin alpha6 and beta4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha6beta4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.     

Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9^+/−;Msx1^+/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.     

Epiprofin/Sp6: a new player in the regulation of tooth development

Odontogenesis is governed by a complex network of intercellular signaling events between the dental epithelium and mesenchyme. This network leads to the progressive determination of tooth shape, and to the differentiation of these tissues into enamel-producing ameloblasts and dentin-producing odontoblasts respectively. Among the main signaling pathways involved in the regulation of tooth development, Bone Morphogenetic Protein (BMP), Sonic hedgehog (Shh) and Wingless-type MMTV integration site (Wnt) pathways have been reported to play significant roles. Recently, the phenotype of mice deficient in Epiprofin/Sp6 (Epfn) has been found to present striking dental abnormalities, including a complete lack of differentiated ameloblasts and consequently no enamel, highly altered molar cusp patterns and the formation of multiple supernumerary teeth. In this article, we review the interaction of Epfn with the BMP, Shh and Wnt pathways in the regulation of tooth development, based on the data obtained from the study of several genetically modified mice.     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

Shh信号通路在牙早期发育中的研究进展

Sonichedgehog（Shh）信号通路在牙早期发育中起关键作用,Shh通过与其特定的受体Ptc／Smo蛋白复合物相互作用来激活整个信号通路。Shh在牙早期发育过程中的表达具有时间和空间特异性,通过自分泌和旁分泌作用于上皮组织以及周围的间充质,促进细胞增殖、分化,调控牙的形态发生。Shh基因缺失将导致小鼠在帽状期牙形态的严重畸形,牙体变小,牙索缺失。对Shh信号通路在牙早期发育的作用及其与Wnt信号通路、BMP家族、FGF家族和MSX家族之间的相互关系进行综述。     

Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina

Vertebrate retinal progenitor cells (RPCs) undergo a robust proliferative expansion to produce enough cells for the retina to form appropriately. Vsx2 (formerly Chx10), a homeodomain protein expressed in RPCs, is required for sufficient proliferation to occur. Sonic Hedgehog protein (SHH), secreted by retinal ganglion cells (RGCs), activates Hedgehog (Hh) signaling in RPCs and is also required for sufficient proliferation to occur. Therefore, we sought to determine if reduced Hh signaling is a contributing factor to the proliferation changes that occur in the absence of Vsx2. To do this, we examined Shh expression and Hh signaling activity in the homozygous ocular retardation J (orJ) mouse, which harbors a recessive null allele in the Vsx2 gene. We found that Shh expression and Hh signaling activity are delayed during early retinal development in orJ mice and this correlates with a delay in the onset of RGC differentiation. At birth, reduced expression of genes regulated by Hh signaling was observed despite the production of SHH ligand. orJ RPCs respond to pre-processed recombinant SHH ligand (SHH-N) in explant culture as evidenced by increased proliferation and expression of Hh target genes. Interestingly, proliferation in the orJ retina is further inhibited by cyclopamine, an antagonist of Hh signaling. Our results suggest that reduced Hh signaling contributes to the reduced level of RPC proliferation in the orJ retina, thereby revealing a role for Vsx2 in mediating mitogen signaling.     

Three-dimensional analysis of mandibular dental root morphology in hominoids

Although often preserved in the fossil record, mandibular dental roots are rarely used for evolutionary studies. This study qualitatively and quantitatively characterizes the three-dimensional morphology of hominoid dental roots. The sample comprises extant apes as well as two fossil species, Khoratpithecus piriyai and Ouranopithecus macedoniensis. The morphological differences between extant genera are observed, quantified and tested for their potential in systematics. Dental roots are imaged using X-ray computerized tomography, conventional microtomography and synchrotron microtomography. Resulting data attest to the high association between taxonomy and tooth root morphology, both qualitatively and quantitatively. A cladistic analysis based on the dental root characters resulted in a tree topology congruent with the consensus phylogeny of hominoids, suggesting that tooth roots might provide useful information in reconstructing hominoid phylogeny. Finally, the evolution of the dental root morphology in apes is discussed.