Msx1 is essential for proper rostral tip formation of the mouse mandible.

The right and left mandibular processes derived from the first branchial arch grow toward the midline and fuse to create the rostral tip region of the mandible during mandibular development. Severe and mild cases of failure in this process results in rare median cleft of the lower lip and cleft chin, respectively. The detailed molecular mechanisms of mandibular tip formation are unknown. We hypothesize that the Msx1 gene is involved in mandibular tip development, because Msx1 has a central role in other craniofacial morphogenesis processes, such as teeth and the secondary palate development. Normal Msx1 expression was observed in the rostral end of the developing mandible; however, a reduced expression of Msx1 was observed in the soft tissue of the mandibular tip than in the lower incisor bud region. The rostral tip of the right and left mandibular processes was unfused in both control and Msx1-null (Msx1-/-) mice at embryonic day (E) 12.5; however, a complete fusion of these processes was observed at E13.5 in the control. The fused processes exhibited a conical shape in the control, whereas the same region remained bifurcated in Msx1-/-. This phenotype occurred with 100% penetrance and was not restored at subsequent stages of development. Furthermore, Meckel's cartilage in addition to the outline surface soft tissues was also unfused and bifurcated in Msx1-/- from E14.5 onward. The expression of phosho-Smad1/5, which is a mediator of bone morphogenetic protein (Bmp) signaling, was downregulated in the mandibular tip of Msx1-/- at E12.5 and E13.5, probably due to the downregulated Bmp4 expression in the neighboring lower incisor bud. Cell proliferation was significantly reduced in the midline region of the mandibular tip in Msx1-/- at the same developmental stages in which downregulation of pSmad was observed. Our results indicate that Msx1 is indispensable for proper mandibular tip development.

Msx1 deficiency interacts with hypoxia and induces a morphogenetic regulation during mouse lip development.

Nonsyndromic clefts of the lip and palate are common birth defects resulting from gene-gene and gene-environment interactions. Mutations in human MSX1 have been linked to orofacial clefting and we show here that Msx1 deficiency causes a growth defect of the medial nasal process (Mnp) in mouse embryos. Although this defect alone does not disrupt lip formation, Msx1-deficient embryos develop a cleft lip when the mother is transiently exposed to reduced oxygen levels or to phenytoin, a drug known to cause embryonic hypoxia. In the absence of interacting environmental factors, the Mnp growth defect caused by Msx1 deficiency is modified by a Pax9-dependent 'morphogenetic regulation', which modulates Mnp shape, rescues lip formation and involves a localized abrogation of Bmp4-mediated repression of Pax9 Analyses of GWAS data revealed a genome-wide significant association of a Gene Ontology morphogenesis term (including assigned roles for MSX1, MSX2, PAX9, BMP4 and GREM1) specifically for nonsyndromic cleft lip with cleft palate. Our data indicate that MSX1 mutations could increase the risk for cleft lip formation by interacting with an impaired morphogenetic regulation that adjusts Mnp shape, or through interactions that inhibit Mnp growth.

Ganoin and acrodin formation on scales and teeth in spotted gar: A vital role of enamelin in the unique process of enamel mineralization.

Gars and bichirs develop scales and teeth with ancient actinopterygian characteristics. Their scale surface and tooth collar are covered with enamel, also known as ganoin, whereas the tooth cap is equipped with an enamel-like tissue, acrodin. Here, we investigated the formation and mineralization of the ganoin and acrodin matrices in spotted gar, and the evolution of the scpp5, ameloblastin (ambn), and enamelin (enam) genes, which encode matrix proteins of ganoin. Results suggest that, in bichirs and gars, all these genes retain structural characteristics of their orthologs in stem actinopterygians, presumably reflecting the presence of ganoin on scales and teeth. During scale formation, Scpp5 and Enam were initially found in the incipient ganoin matrix and the underlying collagen matrix, whereas Ambn was detected mostly in a surface region of the well-developed ganoin matrix. Although collagen is the principal acrodin matrix protein, Scpp5 was detected within the matrix. Similarities in timings of mineralization and the secretion of Scpp5 suggest that acrodin evolved by the loss of the matrix secretory stage of ganoin formation: dentin formation is immediately followed by the maturation stage. The late onset of Ambn secretion during ganoin formation implies that Ambn is not essential for mineral ribbon formation, the hallmark of the enamel matrix. Furthermore, Scpp5 resembles amelogenin that is not important for the initial formation of mineral ribbons in mammals. It is thus likely that the evolution of ENAM was vital to the origin of the unique mineralization process of the enamel matrix.

Ascl1-expressing cell differentiation in initially developed taste buds and taste organoids.

Mammalian taste bud cells are composed of several distinct cell types and differentiated from surrounding tongue epithelial cells. However, the detailed mechanisms underlying their differentiation have yet to be elucidated. In the present study, we examined an Ascl1-expressing cell lineage using circumvallate papillae (CVP) of newborn mice and taste organoids (three-dimensional self-organized tissue cultures), which allow studying the differentiation of taste bud cells in fine detail ex vivo. Using lineage-tracing analysis, we observed that Ascl1 lineage cells expressed type II and III taste cell markers both CVP of newborn mice and taste organoids. However, the coexpression rate in type II cells was lower than that in type III cells. Furthermore, we found that the generation of the cells which express type II and III cell markers was suppressed in taste organoids lacking Ascl1-expressing cells. These findings suggest that Ascl1-expressing precursor cells can differentiate into both type III and a subset of type II taste cells.

Pax9 is required for cardiovascular development and interacts with Tbx1 in the pharyngeal endoderm to control 4th pharyngeal arch artery morphogenesis.

Developmental defects affecting the heart and aortic arch arteries are a significant phenotype observed in individuals with 22q11 deletion syndrome and are caused by a microdeletion on chromosome 22q11. TBX1, one of the deleted genes, is expressed throughout the pharyngeal arches and is considered a key gene, when mutated, for the arch artery defects. Pax9 is expressed in the pharyngeal endoderm and is downregulated in Tbx1 mutant mice. We show here that Pax9-deficient mice are born with complex cardiovascular malformations that affect the outflow tract and aortic arch arteries with failure of the 3rd and 4th pharyngeal arch arteries to form correctly. Transcriptome analysis indicated that Pax9 and Tbx1 may function together, and mice double heterozygous for Tbx1/Pax9 presented with a significantly increased incidence of interrupted aortic arch when compared with Tbx1 heterozygous mice. Using a novel Pax9Cre allele, we demonstrated that the site of this Tbx1-Pax9 genetic interaction is the pharyngeal endoderm, therefore revealing that a Tbx1-Pax9-controlled signalling mechanism emanating from the pharyngeal endoderm is required for crucial tissue interactions during normal morphogenesis of the pharyngeal arch artery system.

Plectin promotes tumor formation by B16 mouse melanoma cells via regulation of Rous sarcoma oncogene activity.

BACKGROUND: Melanoma is a malignant tumor characterized by high proliferation and aggressive metastasis. To address the molecular mechanisms of the proto-oncogene, Rous sarcoma oncogene (Src), which is highly activated and promotes cell proliferation, migration, adhesion, and metastasis in melanoma. Plectin, a cytoskeletal protein, has recently been identified as a Src-binding protein that regulates Src activity in osteoclasts. Plectin is a candidate biomarker of certain tumors because of its high expression and the target of anti-tumor reagents such as ruthenium pyridinecarbothioamide. The molecular mechanisms by which plectin affects melanoma is still unclear. In this study, we examined the role of plectin in melanoma tumor formation. METHODS: We used CRISPR/Cas9 gene editing to knock-out plectin in B16 mouse melanoma cells. Protein levels of plectin and Src activity were examined by western blotting analysis. In vivo tumor formation was assessed by subcutaneous injection of B16 cells into nude mice and histological analysis performed after 2 weeks by Hematoxylin-Eosin (H&E) staining. Cell proliferation was evaluated by direct cell count, cell counting kit-8 assays, cyclin D1 mRNA expression and Ki-67 immunostaining. Cell aggregation and adhesion were examined by spheroid formation, dispase-based dissociation assay and cell adhesion assays. RESULTS: In in vivo tumor formation assays, depletion of plectin resulted in low-density tumors with large intercellular spaces. In vitro experiments revealed that plectin-deficient B16 cells exhibit reduced cell proliferation and reduced cell-to-cell adhesion. Since Src activity is reduced in plectin-deficient melanomas, we examined the relationship between plectin and Src signaling. Src overexpression in plectin knockout B16 cells rescued cell proliferation and improved cell-to-cell adhesion and cell to extracellular matrix adhesion. CONCLUSION: These results suggest that plectin plays critical roles in tumor formation by promoting cell proliferation and cell-to-cell adhesion through Src signaling activity in melanoma cells.

Mash1-expressing cells may be relevant to type III cells and a subset of PLCß2-positive cell differentiation in adult mouse taste buds.

Mammalian taste bud cells have a limited lifespan and differentiate into type I, II, and III cells from basal cells (type IV cells) (postmitotic precursor cells). However, little is known regarding the cell lineage within taste buds. In this study, we investigated the cell fate of Mash1-positive precursor cells utilizing the Cre-loxP system to explore the differentiation of taste bud cells. We found that Mash1-expressing cells in Ascl1CreERT2::CAG-floxed tdTomato mice differentiated into taste bud cells that expressed aromatic L-amino acid decarboxylase (AADC) and carbonic anhydrase IV (CA4) (type III cell markers), but did not differentiate into most of gustducin (type II cell marker)-positive cells. Additionally, we found that Mash1-expressing cells could differentiate into phospholipase C ß2 (PLCß2)-positive cells, which have a shorter lifespan compared with AADC- and CA4-positive cells. These results suggest that Mash1-positive precursor cells could differentiate into type III cells, but not into most of type II cells, in the taste buds.

Msx1 Heterozygosity in Mice Enhances Susceptibility to Phenytoin-Induced Hypoxic Stress Causing Cleft Palate.

OBJECTIVE: Cleft palate is among the most frequent congenital defects in humans. While gene-environment multifactorial threshold models have been proposed to explain this cleft palate formation, only a few experimental models have verified this theory. This study aimed to clarify whether gene-environment interaction can cause cleft palate through a combination of specific genetic and environmental factors. METHODS: Msx1 heterozygosity in mice (Msx1+/-) was selected as a genetic factor since human MSX1 gene mutations may cause nonsyndromic cleft palate. As an environmental factor, hypoxic stress was induced in pregnant mice by administration of the antiepileptic drug phenytoin, a known arrhythmia inducer, during palatal development from embryonic day (E) 11 to E14. Embryos were dissected at E13 for histological analysis or at E17 for recording of the palatal state. RESULTS: Phenytoin administration downregulated cell proliferation in palatal processes in both wild-type and Msx1+/- embryos. Bone morphogenetic protein 4 (Bmp4) expression was slightly downregulated in the anterior palatal process of Msx1+/- embryos. Although Msx1+/- embryos do not show cleft palate under normal conditions, phenytoin administration induced a significantly higher incidence of cleft palate in Msx1+/- embryos compared to wild-type littermates. CONCLUSION: Our data suggest that cleft palate may occur because of the additive effects of Bmp4 downregulation as a result of Msx1 heterozygosity and decreased cell proliferation upon hypoxic stress. Human carriers of MSX1 mutations may have to take more precautions during pregnancy to avoid exposure to environmental risks.

Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate, and Functional Analyses Implicate GREM1 As a Plausible Causative Gene.

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13 × 10(-14) for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32-1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94-1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47-9.61, Pdiff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.

Maldevelopment of the submandibular gland in a mouse model of apert syndrome.

BACKGROUND: Apert syndrome is characterized by craniosynostosis and bony syndactyly of the hands and feet. The cause of Apert syndrome is a single nucleotide substitution mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Clinical experience suggests increased production of saliva by Apert syndrome patients, but this has not been formally investigated. FGFR2 signaling is known to regulate branching morphogenesis of the submandibular glands (SMGs). With the Apert syndrome mouse model (Ap mouse), we investigated the role of FGFR2 in SMGs and analyzed the SMG pathology of Apert syndrome. RESULTS: Ap mice demonstrated significantly greater SMG and sublingual gland (SMG/SLG complex) mass/body weight and percentage of parenchyma per unit area of the SMG compared with control mice. Furthermore, gene expression of Fgf1, Fgf2, Fgf3, Pdgfra, Pdgfrb, Mmp2, Bmp4, Lama5, Etv5, and Dusp6 was significantly higher in the SMG/SLG complex of Ap mice. FGF3 and BMP4 exhibited altered detection patterns. The numbers of macrophages were significantly greater in SMGs of Ap mice than in controls. Regarding functional evaluations of the salivary glands, no significant differences were observed. CONCLUSIONS: These results suggest that the gain-of-function mutation in FGFR2 in the SMGs of Ap mice enhances branching morphogenesis. Developmental Dynamics 247:1175-1185, 2018. © 2018 Wiley Periodicals, Inc.

The transcriptional co-repressor TLE3 regulates myogenic differentiation by repressing the activity of the MyoD transcription factor.

Satellite cells are skeletal muscle stem cells that provide myonuclei for postnatal muscle growth, maintenance, and repair/regeneration in adults. Normally, satellite cells are mitotically quiescent, but they are activated in response to muscle injury, in which case they proliferate extensively and exhibit up-regulated expression of the transcription factor MyoD, a master regulator of myogenesis. MyoD forms a heterodimer with E proteins through their basic helix-loop-helix domain, binds to E boxes in the genome and thereby activates transcription at muscle-specific promoters. The central role of MyoD in muscle differentiation has increased interest in finding potential MyoD regulators. Here we identified transducin-like enhancer of split (TLE3), one of the Groucho/TLE family members, as a regulator of MyoD function during myogenesis. TLE3 was expressed in activated and proliferative satellite cells in which increased TLE3 levels suppressed myogenic differentiation, and, conversely, reduced TLE3 levels promoted myogenesis with a concomitant increase in proliferation. We found that, via its glutamine- and serine/proline-rich domains, TLE3 interferes with MyoD function by disrupting the association between the basic helix-loop-helix domain of MyoD and E proteins. Our findings indicate that TLE3 participates in skeletal muscle homeostasis by dampening satellite cell differentiation via repression of MyoD transcriptional activity.

Nestin expression is differently regulated between odontoblasts and the subodontoblastic layer in mice.

The Nestin gene encodes type VI intermediate filament and is known to be expressed in undifferentiated cells during neurogenesis and myogenesis. To regulate Nestin expression, the first or second intron enhancer is activated in a tissue-dependent manner, for example, the former in mesodermal cells and the latter in neural stem cells. Although Nestin has also been used as a differentiation marker for odontoblasts during tooth development, how Nestin expression is regulated in odontoblasts remains unclear. Therefore, this study aimed to compare the expression patterns of Nestin-GFP (green fluorescent protein) with that of endogenous Nestin in developing teeth of Nestin-EGFP (enhanced GFP) transgenic mice, in which the second intron enhancer is connected with the EGFP domain, at postnatal 7d, 3w, and 8w. Immunohistochemical and in situ hybridization analyses revealed that endogenous Nestin protein and Nestin mRNA were intensely expressed in differentiated odontoblasts, while GFP immunoreactivity, which reflects the activity of Nestin second intron enhancer-mediated transcription, was mainly observed in the subodontoblastic layer. These results indicate that the first intron enhancer may be activated in differentiated odontoblasts. Intriguingly, Nestin-GFP expression in the subodontoblastic layer was found to be restricted to the coronal pulp of molars, which is susceptible to tooth injuries. Because the subodontoblastic layer serves as a reservoir of newly differentiated odontoblast-like cells upon exogenous stimuli to dentin, our findings suggest that the original odontoblasts and regenerated odontoblast-like cells may differently regulate Nestin expression.

SCPP Genes and Their Relatives in Gar: Rapid Expansion of Mineralization Genes in Osteichthyans.

Gar is an actinopterygian that has bone, dentin, enameloid, and ganoin (enamel) in teeth and/or scales. Mineralization of these tissues involves genes encoding various secretory calcium-binding phosphoproteins (SCPPs) in osteichthyans, but no SCPP genes have been identified in chondrichthyans to date. In the gar genome, we identified 38 SCPP genes, seven of which encode "acidic-residue-rich" proteins and 31 encode "Pro/Gln (P/Q) rich" proteins. These gar SCPP genes constitute the largest known repertoire, including many newly identified P/Q-rich genes expressed in teeth and/or scales. Among gar SCPP genes, six acidic and three P/Q-rich genes were identified as orthologs of sarcopterygian genes. The sarcopterygian orthologs of most of these acidic genes are involved in bone and/or dentin formation, and sarcopterygian orthologs of all three P/Q-rich genes participate in enamel formation. The finding of these genes in gar suggests that an elaborate SCPP gene-based genetic system for tissue mineralization was already present in stem osteichthyans. While SCPP genes have been thought to originate from ancient SPARCL1, SPARCL1L1 appears to be more closely related to these genes, because it established a structure similar to acidic SCPP genes probably in stem gnathostomes, perhaps at about the same time with the origin of tissue mineralization. Assuming enamel evolved in stem osteichthyans, all P/Q-rich SCPP genes likely arose within the osteichthyan lineage. Furthermore, the absence of acidic SCPP genes in chondrichthyans might be explained by the secondary loss of earliest acidic genes. It appears that many SCPP genes expanded rapidly in stem osteichthyans and in basal actinopterygians.

Quiescent adult stem cells in murine teeth are regulated by Shh signaling.

The mechanisms regulating the maintenance of quiescent adult stem cells in teeth remain to be fully elucidated. Our aim is to clarify the relationship between BrdU label-retaining cells (LRCs) and sonic hedgehog (Shh) signaling in murine teeth. After prenatal BrdU labeling, mouse pups were analyzed during postnatal day 1 (P1) to week 5 (P5W). Paraffin sections were processed for immunohistochemistry for BrdU, Sox2, Gli1, Shh, Patched1 (Ptch1) and Ki67 and for in situ hybridization for Shh and Ptch1. Dense LRCs, Gli1-(+) cells and Ptch1-(+) cells were co-localized in the outer enamel epithelium of the apical bud and apical dental papilla of incisors. In developing molars, dense LRCs were numerous at P1 but then decreased in number over the course of odontogenesis and were maintained in the center of pulp tissue. Gli1-(+) cells were maintained in the pulp horn during the examined stages, while they increased in number and were maintained in the center of pulp tissue during P2-5W. Ptch1-(+) cells were localized in the pulp horn at P1 and increased in number in the center of the pulp after P3W. Shh mRNA was first expressed in the enamel epithelium and then shifted to odontoblasts and other pulp cells. Shh protein was distributed in the epithelial and mesenchymal tissues of incisors and molars. These findings suggest that quiescent dental stem cells are regulated by Shh signaling, and that Shh signaling plays a crucial role in the differentiation and integrity of odontoblasts during epithelial-mesenchymal interactions and dentinogenesis.

Establishment of in vitro culture system for evaluating dentin-pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells.

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin-pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel's method. We succeeded in establishing an in vitro culture system for evaluating dentin-pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1-3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp-dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.

Allogenic tooth transplantation inhibits the maintenance of dental pulp stem/progenitor cells in mice.

Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.

Ser252Trp mutation in fibroblast growth factor receptor 2 promotes branching morphogenesis in mouse salivary glands.

OBJECTIVES: The purpose of this study was to perform morphological and immunohistochemical (IHC) analysis of the submandibular glands (SMGs) in early development in Apert syndrome model mice (Ap mice). METHODS: ACTB-Cre homozygous mice were mated with fibroblast growth factor receptor 2 (Fgfr2+/Neo-S252W) mice; ACTB-Cre heterozygous mice (ACTB-Cre mice) at embryonic day (E) 13.5 served as the control group, and Fgfr2+/S252W mice (Ap mice) served as the experimental group. Hematoxylin and eosin (H&E) staining was performed on SMGs; Total SMG area and epithelial area were determined, and the epithelial occupancy ratio was calculated. Immunostaining was performed to assess the localization of FGF signaling-related proteins. Next, bromodeoxyuridine (BrdU)-positive cells were evaluated to assess cell proliferation. Finally, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed to assess apoptosis in SMGs. RESULTS: The epithelial occupancy ratio was significantly higher in SMGs of Ap mice compared with that in SMGs of controls. FGF7 and bone morphogenetic protein 4 (BMP4) exhibited different localizations in SMGs of Ap mice compared with SMGs of controls. Cell proliferation was higher in SMGs of Ap mice compared with that of controls; however, apoptosis did not different significantly between the two groups. CONCLUSION: Our results suggest that enhanced FGF signaling conferred by missense mutations in FGFR2 promotes branching morphogenesis in SMGs of Ap mice.

Glucose uptake mediated by glucose transporter 1 is essential for early tooth morphogenesis and size determination of murine molars.

Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1-4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.

Role of osteopontin in the process of pulpal healing following tooth replantation in mice.

Introduction: The role of osteopontin (OPN) following severe injury remains to be elucidated, especially its relationship with type I collagen (encoded by the Col1a1 gene) secretion by newly-differentiated odontoblast-like cells (OBLCs). In this study, we examined the role of OPN in the process of reparative dentin formation with a focus on reinnervation and revascularization after tooth replantation in Opn knockout (KO) and wild-type (WT) mice. Methods: Maxillary first molars of 2- and 3-week-old-Opn KO and WT mice (Opn KO 2W, Opn KO 3W, WT 2W, and WT 3W groups) were replanted, followed by fixation 3-56 days after operation. Following micro-computed tomography analysis, the decalcified samples were processed for immunohistochemistry for Ki67, Nestin, PGP 9.5, and CD31 and in situ hybridization for Col1a1. Results: An intense inflammatory reaction occurred to disrupt pulpal healing in the replanted teeth of the Opn KO 3W group, whereas dental pulp achieved healing in the Opn KO 2W and WT groups. The tertiary dentin in the Opn KO 3W group was significantly decreased in area compared with the Opn KO 2W and WT groups, with a significantly low percentage of Nestin-positive, newly-differentiated OBLCs during postoperative days 7-14. In the Opn KO 3W group, the blood vessels were significantly decreased in area and pulp healing was disturbed with a failure of pulpal revascularization and reinnervation. Conclusions: OPN is necessary for proper reinnervation and revascularization to deposit reparative dentin following severe injury within the dental pulp of erupted teeth with advanced root development.

Exploration of the role of the subodontoblastic layer in odontoblast-like cell differentiation after tooth drilling using Nestin-enhanced green fluorescent protein transgenic mice.

OBJECTIVES: Original odontoblasts and regenerated odontoblast-like cells (OBLCs) may differently regulate Nestin expression. This study aimed to investigate the role of the subodontoblastic layer (SOBL) using green fluorescent protein (GFP) reactivity in the process of OBLC differentiation after tooth drilling in Nestin-enhanced GFP transgenic mice. METHODS: A groove-shaped cavity was prepared on the mesial surface of the maxillary first molars of 5- or 6-week-old mice under deep anesthesia. Immunohistochemical staining for Nestin and GFP and Nestin in situ hybridization were conducted on the sections obtained at 1-14 days postoperative. RESULTS: Odontoblasts showed intense endogenous Nestin protein and mRNA expression, whereas the coronal SOBL cells showed a Nestin-GFP-positive reaction in the control groups. The injured odontoblasts had significantly decreased Nestin immunoreactivity as well as decreased expression of Nestin mRNA 1-2 days after the injury; subsequently, newly differentiated OBLCs were arranged along the pulp-dentin border, with significantly increased Nestin expression as well as increased expression of Nestin mRNA on days 3-5 to form reparative dentin. Nestin-GFP-positive cells at the pulp-dentin border significantly increased in number on days 1 and 2. GFP(+)/Nestin(+) and GFP(-)/Nestin(+) cells were intermingled in the newly differentiated OBLCs. CONCLUSIONS: The commitment of Nestin-GFP-positive cells into Nestin-positive OBLCs suggests that the restriction of endogenous Nestin protein and mRNA expression in the static SOBL cells was removed by exogenous stimuli, resulting in their migration along the pulp-dentin border and their differentiation into OBLCs.