Expression patterns of tenascin-N in the developing mandible

Previous studies have demonstrated that tenascin-N belongs to the family of tenascins, which are found in the extracellular matrix of various embryonic tissues, wounds, and tumors. Tenascin is expressed in the embryonic epithelium, including the neural epithelium from which neural crest cells emerge. However, the expression pattern and role of tenascin-N in the craniofacial region remains unknown. In this study, expression patterns of tenascin-N were confirmed in the mouse craniofacial region from embryonic day 12.5 (E12.5) to postnatal 11. In the diastema region, tenascin-N was strongly expressed in the mesenchyme from E12.5 to E14.5. Tenascin-N expression was also detected in the developing tooth germ. From the bell stage to the premature stage, tenascin- N was expressed in the odontoblasts and ameloblasts of the molar tooth germ, and the ameloblasts of the incisor tooth germ. These findings indicate that the spatial and temporal expression of tenascin-N might have a role in proper mouse craniofacial development, especially tooth developmentope

Pulpal regeneration following allogenic tooth transplantation into mouse maxilla

Autogenic tooth transplantation is now a common procedure in dentistry for replacing a missing tooth. However, there are many difficulties in clinical application of allogenic tooth transplantation because of immunological rejection. This study aims to clarify pulpal regeneration following allogenic tooth transplantation into the mouse maxilla by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) and nestin, and by the histochemistry for tartrate-resistant acid phosphatase (TRAP). The upper right first molar (M1) of 2-week-old mice was extracted and allografted in the original socket in both the littermate and non-littermate after the extraction of M1. Tooth transplantation weakened the nestin-positive reactions in the pulp tissue that had shown immunoreactivity for nestin before operation. On postoperative Days 5-7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in all cases of the littermate group until Day 14, except for one case showing immunological rejection in the pulp chamber. In the non-littermate group, bone-like tissue formation occurred in the pulp chamber in addition to tertiary dentin formation until Day 14. The rate of tertiary dentin was 38%, and the rate of the mixed form of dentin and bone-like tissue formation was 23% (the remainder was immunological rejection). Interestingly, the periodontal tissue recovered even in the case of immunological rejection in which the pulp chamber was replaced by sparse connective tissue. These results suggest that the selection of littermate or non-littermate is decisive for the survival of odontoblast-lineage cells and that the immunological rejection does not influence the periodontal regeneration.ope

Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation

The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.ope

Integument pattern formation involves genetic and epigenetic controls: feather arrays simulated by digital hormone models

Pattern formation is a fundamental morphogenetic process. Models based on genetic and epigenetic control have been proposed but remain controversial. Here we use feather morphogenesis for further evaluation. Adhesion molecules and/or signaling molecules were first expressed homogenously in feather tracts (restrictive mode, appear earlier) or directly in bud or inter-bud regions ( de novo mode, appear later). They either activate or inhibit bud formation, but paradoxically colocalize in the bud. Using feather bud reconstitution, we showed that completely dissociated cells can reform periodic patterns without reference to previous positional codes. The patterning process has the characteristics of being self-organizing, dynamic and plastic. The final pattern is an equilibrium state reached by competition, and the number and size of buds can be altered based on cell number and activator/inhibitor ratio, respectively. We developed a Digital Hormone Model which consists of (1) competent cells without identity that move randomly in a space, (2) extracellular signaling hormones which diffuse by a reaction-diffusion mechanism and activate or inhibit cell adhesion, and (3) cells which respond with topological stochastic actions manifested as changes in cell adhesion. Based on probability, the results are cell clusters arranged in dots or stripes. Thus genetic control provides combinational molecular information which defines the properties of the cells but not the final pattern. Epigenetic control governs interactions among cells and their environment based on physical-chemical rules (such as those described in the Digital Hormone Model). Complex integument patterning is the sum of these two components of control and that is why integument patterns are usually similar but non-identical. These principles may be shared by other pattern formation processes such as barb ridge formation, fingerprints, pigmentation patterning, etc. The Digital Hormone Model can also be applied to swarming robot navigation, reaching intelligent automata and representing a self-re-configurable type of control rather than a follow-the-instruction type of control.ope

Fine tuning of Rac1 and RhoA alters cuspal shapes by remolding the cellular geometry

The anatomic and functional combinations of cusps and lophs (ridges) define the tooth shape of rodent molars, which distinguishes species. The species-specific cusp patterns result from the spatiotemporal induction of enamel knots (EKs), which require precisely controlled cellular behavior to control the epithelial invagination. Despite the well-defined roles of EK in cusp patterning, the determinants of the ultimate cuspal shapes and involvement of epithelial cellular geometry are unknown. Using two typical tooth patterns, the lophodont in gerbils and the bunodont in mice, we showed that the cuspal shape is determined by the dental epithelium at the cap stage, whereas the cellular geometry in the inner dental epithelium (IDE) is correlated with the cuspal shape. Intriguingly, fine tuning Rac1 and RhoA interconvert cuspal shapes between two species by remolding the cellular geometry. Either inhibition of Rac1 or ectopic expression of RhoA could region-distinctively change the columnar shape of IDE cells in gerbils to drive invagination to produce cusps. Conversely, RhoA reduction in mice inhibited invagination and developed lophs. Furthermore, we found that Rac1 and RhoA modulate the choices of cuspal shape by coordinating adhesion junctions, actin distribution, and fibronectin localization to drive IDE invagination.ope

Rgs19 regulates mouse palatal fusion by modulating cell proliferation and apoptosis in the MEE

Palatal development is one of the critical events in craniofacial morphogenesis. During fusion of the palatal shelves, removal of the midline epithelial seam (MES) is a fundamental process for achieving proper morphogenesis of the palate. The reported mechanisms for removing the MES are the processes of apoptosis, migration or general epithelial-to-mesenchymal transition (EMT) through modulations of various signaling molecules including Wnt signaling. RGS19, a regulator of the G protein signaling (RGS) family, interacts selectively with the specific α subunits of the G proteins (Gαi, Gαq) and enhances their GTPase activity. Rgs19 was reported to be a modulator of the Wnt signaling pathway. In mouse palatogenesis, the restricted epithelial expression pattern of Rgs19 was examined in the palatal shelves, where expression of Wnt11 was observed. Based on these specific expression patterns of Rgs19 in the palatal shelves, the present study examined the detailed developmental function of Rgs19 using AS-ODN treatments during in vitro palate organ cultivations as a loss-of-function study. After the knockdown of Rgs19, the morphological changes in the palatal shelves was examined carefully using a computer-aided three dimensional reconstruction method and the altered expression patterns of related signaling molecules were evaluated using genome wide screening methods. RT-qPCR and in situ hybridization methods were also used to confirm these array results. These morphological and molecular examinations suggested that Rgs19 plays important roles in palatal fusion through the degradation of MES via activation of the palatal fusion related and apoptotic related genes. Overall, inhibition of the proliferation related and Wnt responsive genes by Rgs19 are required for proper palatal fusion.ope

Epitope Mapping of Antibodies Suggests the Novel Membrane Topology of B-Cell Receptor Associated Protein 31 on the Cell Surface of Embryonic Stem Cells: The Novel Membrane Topology of BAP31

When located in the endoplasmic reticulum (ER) membrane, B-cell receptor associated protein 31 (BAP31) is involved in the export of secreted proteins from the ER to the plasma membrane. In a previous study, we generated two monoclonal antibodies (mAbs), 297-D4 and 144-A8, that bound to surface molecules on human embryonic stem cells (hESCs), but not to surface molecules on mouse embryonic stem cells (mESCs). Subsequent studies revealed that the mAbs recognized BAP31 on the surface of hESCs. To investigate the membrane topology of BAP31 on the cell surface, we first examined the epitope specificity of 297-D4 and 144-A8, as well as a polyclonal anti-BAP31 antibody (α-BAP31). We generated a series of GST-fused BAP31 mutant proteins in which BAP31 was serially deleted at the C- terminus. GST-fused BAP31 mutant proteins were then screened to identify the epitopes targeted by the antibodies. Both 297-D4 and 144-A8 recognized C-terminal residues 208-217, while α-BAP31 recognized C-terminal residues 165-246, of BAP31 on hESCs, suggesting that the C-terminal domain of BAP31 is exposed on the cell surface. The polyclonal antibody α-BAP31 bound to mESCs, which confirmed that the C-terminal domain of BAP31 is also exposed on the surface of these cells. Our results show for the first time the novel membrane topology of cell surface-expressed BAP31 as the extracellular exposure of the BAP31 C-terminal domain was not predicted from previous studies.ope

Runx3 regulates iron metabolism via modulation of BMP signalling

Objectives: Runx3, a member of the Runx family of transcription factors, has been studied as a tumour suppressor and key player of organ development. In a previous study, we reported differentiation failure and excessive angiogenesis in the liver of Runx3 knock-out (KO) mice. Here, we examined a function of the Runx3 in liver, especially in iron metabolism. Methods: We performed histological and immunohistological analyses of the Runx3 KO mouse liver. RNA-sequencing analyses were performed on primary hepatocytes isolated from Runx3 conditional KO (cKO) mice. The effect of Runx3 knock-down (KD) was also investigated using siRNA-mediated KD in functional human hepatocytes and human hepatocellular carcinoma cells. Result: We observed an iron-overloaded liver with decreased expression of hepcidin in Runx3 KO mice. Expression of BMP6, a regulator of hepcidin transcription, and activity of the BMP pathway were decreased in the liver tissue of Runx3 KO mice. Transcriptome analysis on primary hepatocytes isolated from Runx3 cKO mice also revealed that iron-induced increase in BMP6 was mediated by Runx3. Similar results were observed in Runx3 knock-down experiments using HepaRG cells and HepG2 cells. Finally, we showed that Runx3 enhanced the activity of the BMP6 promoter by responding to iron stimuli in the hepatocytes. Conclusion: In conclusion, we suggest that Runx3 plays important roles in iron metabolism of the liver through regulation of BMP signalling.ope

Periodontal Ligament Cells Are Involved in the Formation of Intracanal Cementum-Like Tissue After Regenerative Endodontic Procedures: A Mouse in situ Model

Regenerative endodontic cell-homing procedures are frequently performed on injured immature teeth diagnosed with pulp necrosis and/or apical periodontitis. The representative histological finding after those procedures is cementum-like tissues filling in the root canal but details of the healing process remain unknown. We investigated that healing process histologically using a mouse in situ model. Regenerative endodontic procedures were experimentally performed on noninfected maxillary first molars of 6-week-old male C57BL/6 mice, after which the healing process was investigated using histology and immunohistochemistry. Immediately after the regenerative endodontic cell-homing procedures, blood clots were seen in the root canals that disappeared over time. On day 7, the blot clot in the root canal was replaced by granulation tissue. From day 14 onward, cementum-like tissues were filled in the root canals, while the amount of fibrous tissue was reduced. Immunohistochemically, positive reactions for periostin were seen in the fibrous tissue in the root canal, the apex, and periodontal ligament cells. On the other hand, positive reactions for nestin were not detected in the root canal. CD31-positive cells with a luminal structure were also observed in the fibrous tissue around the apex and around the newly formed cementum-like tissues in the root canal. Thus, in this study, we have established an in situ mouse model of regenerative endodontic procedures. The results of this study suggest that periodontal ligament cells and vascular endothelial cells grow into the root canals from the apex, replace the blood clots, and participate in the formation of cementum-like tissues with angiogenesis during the healing process of regenerative endodontic procedures.ope

Craniofacial Bone Regeneration using iPS Cell-Derived Neural Crest Like Cells

Induced pluripotent stem (iPS) cells represent a powerful source for cell-based tissue regeneration because they are patient-specific cells and can differentiate into specialized cell types. Previously, we have demonstrated the derivation of neural crest like cells from iPS cells (iPS-NCLCs), and these cells have the potential to differentiate into dental mesenchymal cells, which subsequently differentiate into odontoblasts and dental pulp cells. In this study, we show that iPS-NCLCs can differentiate into mesenchymal stem cells (iPS-NCLC-MSCs), which contribute to craniofacial bone regeneration. iPS-NCLCs were cultured in serum-containing media and differentiated into functional MSCs, as confirmed by expression MSC markers and their ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro. iPS-NCLC-MSCs were negative for markers of undifferentiated iPS cells and did not develop into teratomas when transplanted to immunodeficient mice. Further, iPS-NCLC-MSCs grew normally and differentiated into osteoblasts on hydroxyapatite scaffolds in vitro. To assess the potential of iPS-NCLC-MSCs to regenerate craniofacial bone in vivo, iPS-NCLC-MSCs were transplanted into critical-size calvarial defects in immunodeficient mice for 8 weeks. Histological analysis revealed that iPS-NCLC-MSCs differentiated into osteoblasts and contributed to bone regeneration without tumor formation. These results indicate that iPS-NCLC-MSCs could be a potential candidate for cell-based craniofacial bone tissue repair and regeneration.ope