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Enamel knot (EK)—a signaling center—refers to a transient morphological structure comprising epithelial tissue. EK is believed to regulate tooth development in early organogenesis without its own cellular alterations, including proliferation and differentiation. EKs show a very simple but conserved structure and share functions with teeth of recently evolved vertebrates, suggesting conserved signaling in certain organs, such as functional teeth, through the course of evolution. In this study, we examined the expression patterns of key EK-specific genes including Dusp26 , Fat4, Meis2, Sln , and Zpld1 during mice embryogenesis. Expression patterns of these genes may reveal putative differentiation mechanisms underlying tooth morphogenesis.

miRNAs are conserved short non-coding RNAs that play a role in the modulation of various biological pathways during tissue and organ morphogenesis. In this study, the function of miRNA-221-3p in tooth development, through its loss or gain in function was evaluated. A variety of techniques were utilized to evaluate detailed functional roles of miRNA-221-3p during odontogenesis, including in vitro tooth cultivation, renal capsule transplantation, in situ hybridization, real-time PCR, and immunohistochemistry. Two-day in vitro tooth cultivation at E13 identified altered cellular events, including cellular proliferation, apoptosis, adhesion, and cytoskeletal arrangement, with the loss and gain of miRNA-221-3p. qPCR analysis revealed alterations in gene expression of tooth-related signaling molecules, including β-catenin, Bmp2, Bmp4, Fgf4, Ptch1, and Shh, when inhibited with miRNA-221-3p and mimic. Also, the inhibition of miRNA-221-3p demonstrated increased mesenchymal localizations of pSMAD1/5/8, alongside decreased expression patterns of Shh and Fgf4 within inner enamel epithelium (IEE) in E13 + 2 days in vitro cultivated teeth. Moreover, 1-week renal transplantation of in vitro cultivated teeth had smaller tooth size with reduced enamel and dentin matrices, along with increased cellular proliferation and Shh expression along the Hertwig epithelial root sheath (HERS), within the inhibitor group. Similarly, in 3-week renal calcified teeth, the overexpression of miRNA-221-3p did not affect tooth phenotype, while the loss of function resulted in long and slender teeth with short mesiodistal length. This study provides evidence that a suitable level of miRNA-221-3p is required for the modulation of major signaling pathways, including Wnt, Bmp, and Shh, during tooth morphogenesis.

The function and mechanism of action of MLL-TET1 (MT1) fusion protein in hematological cells are unclear and require further investigation. In the present study, we found that the MT1 fusion protein attenuated the expression of Cebpa, Csf1r, and Cd11b and inhibited the differentiation of myeloid progenitor cells. Increased binding of the MT1 fusion protein to the Trib2 promoter upregulated Trib2 mRNA and protein expression and downregulated Cebpa expression. Trib2 knockdown relieved the inhibition of myeloid cell differentiation induced by the MT1 fusion protein. Thus, TRIB2 is important for the survival of leukemia cells during MT1-related leukemogenesis and is important in maintaining differentiation blockade of leukemic cells. KEY MESSAGES: • MLL-TET1 fusion decreases the 5-hmC levels in the myeloid progenitor cells. • MLL-TET1 fusion inhibits myeloid differentiation through decreased expression of Cebpa. • MLL-TET1 fusion blocks the differentiation of the myeloid progenitor cells by overexpressing Trib2. • Knockdown of Trib2 in MLL-TET1 transduced cells induces myeloid differentiation.

Objective To evaluate the effect of resveratrol on periodontal bone regeneration after local delivery and to determine its effect on inflammatory mediators. Background Resveratrol is considered an anti-inflammatory polyphenolic stilbene involved in the modulation of inflammation. Materials and methods Periodontitis was induced in mouse molars using a 5-day ligature model followed by the left second molar extraction and 50 µM resveratrol treatment for 1 and 2 weeks. We then examined specimens treated for 1 week histologically and with immunostaining. Microfocus-computed tomography (micro-CT) was used to examine the bone volume formation. Results After 1 week of treatment, proinflammatory cytokine levels (TNF-alpha and IL6), cells exhibiting neutrophil and macrophage marker (MPO), cell proliferation marker (Ki67), and preosteoblastic marker (RUNX2) reactivity decreased in the resveratrol-treated specimens compared to the control group. In contrast, we observed a higher number of CD31-, F4/80-, and osteocalcin- (OCN-) positive cells in the resveratrol-treated specimens. After 2 weeks, micro-CT confirmed an increased bone mass in the region of the extraction socket in the resveratrol-treated group. Conclusion After 1 week, the resveratrol-treated specimens revealed evidence of inflammation modulation compared to the control group. These data suggest that resveratrol not only affects inflammation control but also is useful for treating periodontitis-related tissue defects and bone regeneration.

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson&rsquo, s trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including &alpha, SMA, CK14, IL-1&beta, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

Circumvallate papilla (CVP) is a distinctively structured with dome-shaped apex, and the surrounding trench which contains over two hundred taste buds on the lateral walls. Although CVP was extensively studied to determine the regulatory mechanisms during organogenesis, it still remains to be elucidated the principle mechanisms of signaling regulations on morphogenesis including taste buds formation. The key role of Yes-associated protein (YAP) in the regulation of organ size and cell proliferation in vertebrates is well understood, but little is known about the role of this signaling pathway in CVP development. We aimed to determine the putative roles of YAP signaling in the epithelial patterning during CVP morphogenesis. To evaluate the precise localization patterns of YAP and other related signaling molecules, including β-catenin, Ki67, cytokeratins, and PGP9.5, in CVP tissue, histology and immunohistochemistry were employed at E16 and adult mice. Our results suggested that there are specific localization patterns of YAP and Wnt signaling molecules in developing and adult CVP. These concrete localization patterns would provide putative involvements of YAP and Wnt signaling for proper epithelial cell differentiation including the formation and maintenance of taste buds.

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs that post-transcriptionally regulate gene expression in organisms. Most mammalian miRNAs influence biological processes, including developmental changes, tissue morphogenesis and the maintenance of tissue identity, cell growth, differentiation, apoptosis, and metabolism. The miR-206-3p has been correlated with cancer, however, developmental roles of this miRNA are unclear. In this study, we examined the expression pattern and evaluated the developmental regulation of miR-206-3p during tooth morphogenesis using ex-vivo culture method. The expression pattern of miR-206-3p was examined in the epithelium and mesenchyme of developing tooth germ with stage-specific manners. Perturbation of the expression of miR-206-3p clearly altered expression patterns of dental-development&ndash, related signaling molecules, including Axin2, Bmp2, Fgf4, Lef1 and Shh. The gene expression complemented with change in cellular events including, apoptosis and proliferation which caused altered crown and pulp morphogenesis in renal-capsule&ndash, calcified teeth. Especially, mislocalization of &beta, Catenin and SMAD1/5/8 were observed alongside dramatic alterations in the expression patterns of Fgf4 and Shh. Overall, our data suggest that the miR-206-3p regulate the cellular physiology during tooth morphogenesis through modulation of the Wnt, Bmp, Fgf, and Shh signaling pathways to form proper tooth pulp and crown.

The endoplasmic reticulum (ER) is a site where protein folding and posttranslational modifications occur, but when unfolded or misfolded proteins accumulate in the ER lumen, an unfolded protein response (UPR) occurs. A UPR activates ER-stress signalling genes, including inositol-requiring enzyme-1 (Ire1), activating transcription factor 6 (Atf6), and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (Perk), to maintain homeostasis. The involvement of ER stress molecules in metabolic disease and hard tissue matrix formation has been established; however, an understanding of the role of ER-stress signalling molecules in tooth development is lacking. The aims of this study are to define the stage-specific expression patterns of ER stress-related molecules and to elucidate their putative functions in the organogenesis of teeth. This study leverages knowledge of the tissue morphology and expression patterns of a range of signalling molecules during tooth development. RT-qPCR, in situ hybridization, and immunohistochemistry analyses were performed to determine the stage-specific expression patterns of ER-stress-related signalling molecules at important stages of tooth development. RT-qPCR analyses showed that Atf6 and Perk have similar expression levels during all stages of tooth development; however, the expression levels of Ire1 and its downstream target X-box binding protein (Xbp1) increased significantly from the cap to the secretory stage of tooth development. In situ hybridization results revealed that Atf6 and Xbp1 were expressed in cells that form the enamel knot at cap stage and ameloblasts and odontoblasts at secretory stage in stage-specific patterns. In addition, Atf6, Ire1, and Xbp1 expression exhibited distinct localization patterns in secretory odontoblasts and ameloblasts of PN0 molars. Overall, our results strongly suggest that ER-stress molecules are involved in tooth development in response to protein overload that occurs during signaling modulations from enamel knots at cap stage and extracellular matrix secretion at secretory stage.

In mice, tongue epithelial differentiation is mainly regulated by the interactions among various signalling molecules including Fgf signalling pathways. However, the subsequent signalling modulations for epithelial maturation, initiated by Fgf signalling, remain to be elucidated. Therefore, we employed an in vitro tongue organ cultivation system along with the applications of various pharmacological inhibitors against the intracellular signalling molecules of Fgf signalling pathways, including H89, LY294002, PD98059, and U0126. Following treatments with LY294002 and H89, inhibitors for PI3K and PKA, respectively, the decreased thickness of the tongue epithelium was observed along with the alteration in cell proliferative and apoptotic patterns. Meanwhile, cultivated tongues treated with MEK inhibitor U0126 or PD98059 showed significantly decreased cell proliferation in the tongue epithelium and the mesenchyme. Based on these results, we suggest that the tongue epithelium is differentiated into multiple epithelial cell layers via the PI3K and PKA pathways in tissue-specific manner during the epithelial-mesenchymal interactions.

Objectives Chronic injury in tongue causes the variety of reactions in the oral cavity, frequently leading to its functional and structural disintegrity including inflammation and sensory dysfunction, but its detailed profiles were not elucidated yet. One of the chronically injured tongue such as tongue piercing, as a pathological aspect, is currently popular among younger people but may be associated with severe side effects, leading to pathophysiological complications. However, the pathophysiological aspects and related cellular and molecular mechanisms underlying tongue injury are not clearly understood. Design In this study, we designed an experimental model system using C57BL/6 male mice that mimics a chronically injured situation by penetrating the middle part of tongue with silk suture. After 5 and 10 days mice were sacrificed and tongues were collected and processed for histological evaluation and immunohistochemistry. Results We found that the anterior tongue showed localization of neuro-inflammatory signaling molecules such as myeloperoxidase (MPO), matrix metalloproteinase 2 (MMP2), tumor necrosis factor-α (TNF-α), nerve growth factor, and transient receptor potential cation channel subfamily V member 1 (TRPV1) without any apparent inflammation in temporal manner. In addition, the signal for AM1-43, an activity-dependent nerve terminal probe, decreased within the fungiform papillae on the anterior tongue after injury. Conclusions These results implied that the distinct localizations of inflammatory cytokines and neurotrophin would contribute altered sensory function in anterior tongue following the chronic injury. Our study indicates the possible pathophysiologic mechanism underlying neuro-inflammation following chronically injury of tongue. In addition, it could be cautiously postulated that mechanical injury should be avoided to prevent chronic pain disorders from being triggered.

Objective We evaluated the role of oleanolic acid acetate (OAA), a triterpenoid commonly used in the treatment of liver disorders, inflammatory diseases, and metastasis, in bone formation after tooth loss by periodontitis. Background Periodontitis causes the sequential degradation of the alveolar bone and associated structures, resulting in tooth loss. Several studies have attempted to regenerate the bone for implantation following tooth loss. Methods Maxillary left second molar was extracted from 8-week-old male mice following induction of periodontitis by ligature for 5 days. The extraction socket was treated with 50 ng/µL OAA for 1, 2, and 3 weeks. Detailed morphological changes were examined using Masson's trichrome staining, and the precise localization patterns of various signaling molecules, including CD31, F4/80, interleukin (IL)-6, and osteocalcin, were observed. The volume of bone formation was examined by Micro-CT. Osteoclasts were enumerated using tartrate-resistant acid phosphatase (TRAP) staining. For molecular dissection of signaling molecules, we employed the hanging-drop in vitro cultivation method at E14 for 1 day and examined the expression pattern of transforming growth factor (TGF)-β superfamily and Wnt signaling genes. Results Histomorphometrical examinations showed facilitated bone formation in the extraction socket following OAA treatment. In addition, OAA-treated specimens showed the altered localization patterns of inflammatory and bone formation-related signaling molecules including CD31, F4/80, IL-6, and osteocalcin. Also, embryonic tooth germ mesenchymal tissue cultivation with OAA treatment showed the significant altered expression patterns of signaling molecules such as transforming growth factor (TGF)-β superfamily and Wnt signaling. Conclusions Oleanolic acid acetate induces bone formation and remodeling through proper modulation of osteoblast, osteoclast, and inflammation with regulations of TGF-β and Wnt signaling.

Salivary gland (SG) development involves dynamic epithelial-mesenchymal interactions resulting in the formation of highly branched epithelial structures that produce and secrete saliva. The SG epithelium differentiates into saliva-producing terminal buds, i.e., acini, and transporting ducts. Most studies on the salivary gland have focused on branching morphogenesis; however, acinar cell differentiation underlying the determination of serous or mucous salivary glands is unclear. The objective of this study was to identify the mesenchymal signaling molecules involved in the epithelial differentiation of the salivary gland type as serous or mucous. Salivary glands undergoing stage-specific development, including the parotid gland (PG) and the sublingual gland (SLG) at embryonic day 14.5 (E14.5) were dissected. The glands were treated with dispase II to separate the epithelium and the mesenchyme. RNA from mesenchyme was processed for microarray analysis. Thereafter, microarray data were analyzed to identify putative candidate molecules involved in salivary gland differentiation and confirmed via quantitative reverse transcription polymerase chain reaction. The microarray analysis revealed the expression of 31,873 genes in the PG and SLG mesenchyme. Of the expressed genes 21,026 genes were found to be equally expressed (Fold change 1.000) in both PG and SLG mesenchyme. The numbers of genes expressed over onefold in the PG and SLG mesenchyme were found to be 5247 and 5600 respectively. On limiting the fold-change cut off value over 1.5 folds, only 214 and 137 genes were expressed over 1.5 folds in the PG and the SLG mesenchyme respectively. Our findings suggest that differential expression patterns of the mesenchymal signaling molecules are involved in fate determination of the salivary acinar cell types during mouse embryogenesis. In the near future, functional evaluation of the candidate genes will be performed using gain- and loss-of-function mutation studies during in vitro organ cultivation.

Epithelial differentiation is thought to be determined by mesenchymal components during embryogenesis. In mice, palatal mucosa showed the region-specific keratinization pattern along antero-posterior axis. However, developmental mechanisms involved in oral mucosa differentiation with fine tuning of keratinization are not elucidated yet. To reveal this developmental mechanism, first, we conducted tissue recombination assay of the palate at E16 for 2 days which revealed that epithelial differentiation with specific localization of CK10 is modulated by mesenchymal components. Based on the results, we propose that mesenchymal signaling would determine the presumptive fate of developing palatal epithelium in spatiotemporal manner. Genome-wide screening analysis using laser micro-dissection to collect spatiotemporal specific molecules between anterior and posterior palate suggested Meox2 in the posterior mesenchymal tissue to be a candidate regulator controlling epithelial differentiation. To examine the detailed spatiotemporal function of Meox2, we employed in vitro organ cultivation with the loss- and gain-of-function studies at E14.5 for 2 and 4 days, respectively. Our results suggest that posteriorly expressed Meox2 modulates non-keratinized epithelial differentiation through complex signaling regulations in mice palatogenesis.

Adenomatosis polyposis coli downregulated 1 (APCDD1), a negative regulator of Wnt signaling, was examined to understand detailed mechanisms underlying Wnt signaling tooth development. In situ hybridization showed that Apcdd1 was expressed in the condensed mesenchyme at the bud stage, and in the inner enamel epithelium (IEE), including enamel knot (EK) at the cap stage. In vitro organ cultivation by using Apcdd1 antisense oligodeoxynucleotides was performed at E13.5 for 2 days to define the developmental functions of APCDD1 during tooth development. Analysis of histogenesis and cellular events such as cell adhesion, proliferation, apoptosis and epithelial rearrangement after Apcdd1 knockdown showed altered morphogenesis of the tooth germ with decreased cell proliferation and altered localization of cell adhesion molecules. Actin filament staining and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) labeling of IEE cells showed that Apcdd1 knockdown enhanced epithelial rearrangement in the IEE and EK. To understand the precise signaling regulations of Apcdd1, we evaluated the altered expression patterns of signaling molecules, related with Wnt and enamel knot signalings using RT-qPCR. Tooth germs at cap stage were transplanted into the kidney capsules and were allowed to develop into calcified teeth for 3 weeks. Apcdd1 knockdown increased the number of ectopic cusps on the mesial side of the tooth. Our results suggested that APCDD1 modulates the gene expression of Wnt- and EK-related signaling molecules at the cap stage of tooth development, and is involved in tooth cusp patterning by modulating the epithelial rearrangement in the IEE.

After palatal fusion, the dorsal and ventral epithelia of the palatal shelf differentiate into the nasal and oral mucosa, respectively. The tissue-specific differentiation of palatal epithelia along the dorsal-ventral axis is regulated by the signaling molecules expressed in the underlying mesenchyme. Thus, as in many other epithelial organs, differentiation relies on epithelial-mesenchymal interactions. To screen for region-specific mesenchymal signaling molecules that determine the fate of the palatal epithelia, we employed a laser microdissection (LMD) method. LMD allowed us to collect region-specific mesenchymal tissues at E13, prior to palatal fusion and the development of distinct dorsal and ventral epithelial morphology. Genome-wide screening was performed on the tissues collected using LMD to identify candidate mesenchymal signaling molecules. The microarray results were validated using real-time quantitative (qPCR) and in situ hybridization methods. The developmental role and interactions of the candidate genes were evaluated in in vitro-cultivated E13 palates using an anti-sense oligodeoxynucleotide (AS-ODN)-based loss-of-function approach. Apparent changes in the expression patterns of Runt-related transcription factor 2 (Runx2) and LIM homeobox 8 (Lhx8) were observed after knocking down each gene. Knock-down of Runx2 and Lhx8 also altered the immunolocalization pattern of cytokeratin18 (CK18), an established marker for nasal epithelium. These results were confirmed using Runx2 heterozygote mice. The mesenchymal signaling molecules Runx2 and Lhx8, which possess region-specific expression patterns along the dorsoventral axis, functionally interact to regulate the cellular and molecular characteristics of dorsal and ventral epithelia, suggesting that mesenchymal signaling molecules determine the dorsoventral fate of epithelial structures in the developing palate.

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.

The alveolar bone process is the thickened ridge of bone that bears the teeth and is known to have dynamic functional interactions with surrounding tissues. However, the detailed morphological changes that occur during alveolar bone process development and the underlying molecular mechanisms behind this morphogenesis have not been elucidated. In this study, we examined the detailed morphological changes of the alveolar bone process during mouse development using HE and MTC staining. In addition, we evaluated the precise localization pattern of various signaling molecules involved in blood vessel formation including CD31, α-SMA, VEGF, periostin, and TGF-β. Innervation of the alveolar bone process was examined following injection of the nerve terminal dye AM1-43. The morphological and immunohistochemical data suggested that there is an intimate relationship between alveolar bone process development and blood vessel formation. To more closely examine the role of blood vessels in alveolar bone process formation, we microinjected mice with a clinically available anti-VEGF antibody, bevacizumab, at PN5 and analyzed the effects 5 days later. Compared to the control animals, anti-VEGF treated animals showed a disruption of the integration of bony tissues to form the alveolar bone process structures, which should contain the periodontal ligaments. Based on these data, we conclude that specific morphogenesis of the alveolar bone process is closely associated with blood vessel formation.

Teraspanin transmembrane protein, Perp (P53 apoptosis effector related to PMP22), which is found in the plasma membrane as a component of the desmosome, is reported to be involved in the morphogenesis of the epithelium and the enamel formation of the incisor. However, its expression pattern and signaling regulation during molar development have not been elucidated in detail. We have examined the precise expression patterns of Perp in developing lower molars and employed the knock-down of Perp by antisense oligodeoxynucleotide treatment during in vitro organ cultivation at embryonic day 13 to define the precise developmental function of Perp. Perp was expressed mainly in the dental lamina and stellate reticulum regions at the bud and cap stages. After Perp knock-down, the tooth germ showed disruption of the dental lamina and stellate reticulum with altered apoptosis and proliferation. The changed expression levels of related signaling molecules from the enamel knot and desmosome were evaluated by real-time quantitative polymerase chain reaction. A renal capsule transplantation method was employed to examine the effects of Perp knock-down on molar crown development. Ultrastructural observations revealed that enamel was deposited more densely in an irregular pattern in the cusp region, and that dentin was hypo-mineralized after Perp knock-down at the cap stage. Thus, Perp might play important roles in the formation and integration of stellate reticulum, dental lamina structure and enamel formation through signaling interactions with the enamel knot and desmosome-related signaling molecules at the cap stage of lower molar development.

An in vitro culture model enabling posterior capsule opacification (PCO) to be investigated was developed and established by using low-melting-point (LMP)-agarose gel to support the capsular bag. After removal of the cornea from rodent and porcine eyeballs, the lens zonules were dissected. Whole lens explants were embedded into 2 % (37 °C) LMP-agarose gel solution. As performed routinely in cataract surgery, capsulotomy and lens fiber removal were carried out in the solidified LMP-agarose gel as sham cataract surgery. The LMP-agarose-gel-supported capsular bag/lens epithelial cell (CB-LEC) complexes were maintained in Dulbecco's modified Eagle medium supplemented with 10 % fetal bovine serum in an anterior face-down position. The proliferation and migration of LECs into the posterior capsule were observed every 12 h by phase-contrast microscopy. Epithelial cells were observed at the central portion of the CB-LEC complexes after 56.57 ± 16.56 h (n = 7) and 106 ± 14.03 h (n = 6) of culture, for rodent and porcine lenses, respectively. The solidified gel allowed clear microscopic observations and whole-mount immunostaining evaluations of the whole area of the capsular bag. Histological examinations revealed the proliferation, migration, and transdifferentiation of LECs related to posterior capsule opacification. This new in vitro culture model provides experimental benefits by maintaining the natural contour of the capsule without implants inside or outside of the capsule. In addition, this model system allows pharmacological and histological evaluations of the cultured CB-LEC complexes without additional manipulations.

Grainyhead-like 3 (Grhl3) is a transcription factor involved in epithelial morphogenesis. In the present study, we evaluated the developmental role of Grhl3 in structural formation of the circumvallate papilla (CVP), which undergoes dynamic morphological changes during organogenesis. The specific expression pattern of Grhl3 was examined in the CVP-forming region, specifically in the apex and epithelial stalk from E13.5 to E15.5 using in situ hybridization. To determine the role of Grhl3 in epithelial morphogenesis of the CVP, we employed an in vitro tongue culture method, wherein E13.5 tongue were isolated and cultured for 2 days after knocking down of Grhl3. Knockdown of Grhl3 resulted in significant changes to the epithelial structure of the CVP, such that the apical region of the CVP was smaller in size, and the epithelial stalks were more deeply invaginated. To define the mechanisms underlying these morphological alterations, we examined cell migration, proliferation, and apoptosis using phalloidin staining, immunohistochemistry against Ki67, ROCK1, and E-cadherin, and a TUNEL assay, respectively. These results revealed an increase in proliferation, a reduction in apoptosis, and an altered pattern of cytoskeletal formation in the CVP-forming epithelium, following Grhl3 knockdown. In addition, there were changes in the specific expression patterns of signaling and apoptosis-related molecules such as Axin2, Bak1, Bcl2, Casp3, Casp8, Ctnnb1, Cnnd1, Gli3, Lef1, Ptch1, Rock1, Shh, and Wnt11, which could explain the altered cellular and morphological events. Based on these results, we propose that developmental stage-specific Grhl3 plays a significant role in CVP morphogenesis not by just disruption of epithelial integrity but by regulating epithelial cell proliferation, apoptosis, and migration via Shh, Wnt, and apoptosis signaling during mouse embryogenesis.

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.

Different from humans, who have a continuous dentition of teeth, mice have only three molars and one incisor separated by a toothless region called the diastema in the hemi mandibular arch. Although tooth buds form in the embryonic diastema, they regress and do not develop into teeth. In this study, we evaluated the proteins that modulate the diastema formation through comparative analysis with molar-forming tissue by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteome analysis. From the comparative and semi-quantitative proteome analysis, we identified 147 up- and 173 down-regulated proteins in the diastema compared to the molar forming proteins. Based on this proteome analysis, we selected and evaluated two candidate proteins, EMERIN and RAB7A, as diastema tissue specific markers. This study provides the first list of proteins that were detected in the mouse embryonic diastema region, which will be useful to understand the mechanisms of tooth development [BMB Reports 2012; 45(6): 337-341]

The cell-fate determination factor Dachshund, a component of the Retinal Determination Gene Network (RDGN), has a role in breast tumor proliferation through the repression of cyclin D1 and several key regulators of embryonic stem cell function, such as Nanog and Sox2. However, little is known about the role of DACH1 in a myeloid lineage as a cell cycle regulator. Here, we identified the differential expression levels of extensive cell cycle regulators controlled by DACH1 in myeloid progenitor cells. The forced expression of DACH1 induced p27(Kip1) and repressed p21(Cip1), which is a pivotal characteristic of the myeloid progenitor. Furthermore, DACH1 significantly increased the expression of cyclin D1, D3, F, and Cdk 1, 4, and 6 in myeloid progenitor cells. The knockdown of DACH1 blocked the cell cycle progression of HL-60 promyeloblastic cells through the decrease of cyclin D1, D3, F, and Cdk 1, 4, and 6 and increase in p21(Cip1), which in turn decreased the phosphorylation of the Rb protein. The expression of Sox2, Oct4, and Klf4 was significantly up-regulated by the forced expression of DACH1 in mouse myeloid progenitor cells.

Epithelial appendages on palatal rugae develop during mouse palatogenesis through epithelial thickening and pattern formation. Recently, the patterned formation of nine rugae was observed together with the specific expression patterns of Shh in rodents. However, no crucial evidence was found for a direct association between Shh expression and the distinct structural formation of rugae. In order to reveal possible relationships, we investigated the morphological changes of rugae and expression patterns of Shh directly by in vitro organ culture at embryonic day 13 (E13) for 2 days. To compare and examine the diverse growing aspects of the palate and rugae, we carefully observed the detailed morphogenesis, with cell proliferation of the rugae occurring between E13 and E14.5. After 2 days of cultivation at E13, DiI micro-injections revealed that the middle part of the palate, adjacent to the upper molar-forming region, contributed to the formation of the subsequent structure of rugae by extensive cell rearrangement and proliferation within the epithelium in the preferred anteroposterior direction. The results also defined the intimate relationship between Shh expression and rugae formation.

During palatine rugae development in mice, a cell cluster appeared transiently in the palatine rugae epithelium. The characteristics of these cells were examined histologically and immunohistochemically for anti-cytokeratin (CK) and anti-protein gene product 9.5 (PGP9.5) antibodies using mice from embryonic day 15 (E15) to postnatal day 1 (PN1). At E15, the epithelium of both palatine rugae located in the anterior portion and inter-rugae area consisted of 2-3 cell layers, and palatine rugae have papillae under the epithelium. However, palatine rugae located in the posterior portion have cell clusters in the epithelium. These cells expressed immunopositive reactions for anti-CK18, although they did not react with anti-CK14. Under the anti-CK18 immunopositive cell cluster in palatine rugae epithelium, anti-PGP9.5 immunoreactivity was observed in papillae. Moreover, the basal cells of the cell cluster showed anti-CK7 immunoreactivity. The cell cluster in palatine rugae epithelium disappeared gradually and anti-CK18 immunopositive cells also disappeared. However, anti-PGP9.5 immunoreactivity in the papilla was maintained after the disappearance of anti-CK18 immunoactivity in the palatine rugae epithelium. At PN1, the epithelium of the palate, including the palatine rugae, was stratified and keratinized. These findings suggested that the cell cluster in the palatine rugae epithelium may be considered as a taste bud-like structure according to the expression pattern for anti-CK and may play an important role in nerve innervation to the papilla under the palatine rugae epithelium.

Oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-DNA) have gained attention as potentially useful therapeutics. However, the phosphorothioate-modified CpG-DNAs (PS-ODN) can induce backbone-related side effects. Here, we compared the immunostimulatory activity of natural phosphodiester CpG-DNA (PO-ODN) from Mycobacterium bovis and PS-ODN in mice. Both PO-ODN and PS-ODN induced production of IL-12. PS-ODN increased spleen weights, spleen cell numbers, and the migration of macrophages into the peritoneal cavity in the mice in a CG sequence-dependent manner. PS-ODN induced anti-PS-ODN antibody production in the mice, and the PS-ODN-specific IgM was cross-reactive with other PS-ODNs in a CG sequence-independent manner. In contrast, PO-ODN did not affect on spleen weights, cell numbers, or IgM production. These results may provide an explanation for the side effects in immunotherapeutic application of PS-ODN. They also suggest that PO-ODN may be more optimal than PS-ODN to enhance innate immune responses without severe side effects.

Schlafen-2 (slfn-2) is a member of slfn family, regulators of T cell development and its expression is altered during infection by microbial pathogens. However, the molecular mechanism involved in slfn expression is still to be determined. In this study, we isolated slfn-2 as a LPS-induced differentially expressed genes (DEGs) in RAW 264.7 cells and examined expression and regulation of slfn-2 in CpG-DNA-treated and LPS-treated macrophages. We defined a transcriptional start site in the slfn-2 gene. To examine the promoter organization of the slfn-2 gene, we cloned a approximately 1.8 kb region upstream of the transcription start site. Sequence analysis indicates consensus sites for AP-1 and NF-kappaB. Comprehensive mutant analyses, ELISA-based transcription factor activation assay, and ChIP assays reveal that functional interaction of AP-1 and NF-kappaB with the promoter element is necessary for the Toll-like receptor (TLR)-mediated slfn-2 gene expression by CpG-DNA and LPS treatment in macrophages. In summary, we identified a slfn-2 promoter for the first time and demonstrated that CpG-DNA and LPS triggers slfn-2 gene expression by activating NF-kappaB and AP-1 pathways in macrophages.

Synthetic oligodeoxynucleotides (CpG-ODNs) and bacterial DNA containing unmethylated CpG dinucleotides in the context of particular base sequences (CpG motifs) are known to inhibit anti-IgM-induced growth arrest and apoptosis of WHEI 231 B lymphocytes, and spontaneous apoptosis of mature spleen B cells in a sequence-specific fashion of the CpG-ODN. Here we report that CpG-ODN protects from the cell death induced by γ-irradiation of primary mouse spleen cells as well as mouse RAW 264.7 macrophage cells and human RPMI 8226 B cells. Experimental results showed that CpG-ODN promotes growth of the cells, and protects the cells from γ-irradiation-induced cell death accompanying Bcl-xS/L and Bcl-2 upregulation. Furthermore, survival of macrophages was enhanced when splenocytes were pretreated with CpG-ODN. Our results suggest the potential application of CpG-ODNs for more efficient cancer radiotherapy by enhancing survival of normal immune cells after radiation damage.

Pyrrolidine dithiocarbamate (PDTC) is a stable compound that acts as antioxidant or prooxidant, and is widely used to inhibit the activation of NF-kappaB. PDTC was also reported to activate NF-kappaB depending on its dose and metal ions in PC12 cells. In this work, we demonstrated a working mechanism of PDTC and its effects on the proinflammatory cytokine gene expression in a mouse macrophage cell line, RAW 264.7. PDTC alone induced NF-kappaB-independent MIP-2 promoter activation that can be assessed by transient transfection and confocal image analysis. The involvement of AP-1 transcription factor was noticed by promoter deletion/site-specific mutation analysis and electrophoretic mobility shift assay (EMSA). Among three different mitogen-activated protein kinase (MAPK) pathways tested, only the stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathway was significantly activated in RAW 264.7 cells after the stimulation with PDTC. Using pathway-specific inhibitors, we found that the SAPK/JNK pathway is clearly associated with PDTC-induced MIP-2 gene expression. Our experimental results indicate that PDTC-induced proinflammatory cytokine expressions are mediated by SAPK/JNK pathway, which activates AP-1.

The cytomegalovirus immediate-early (CMV IE) gene enhancer/promoter regulates the expression of immediate-early gene products and initiation of CMV replication. TNF-alpha and lipopolysaccharide (LPS) strongly activate the promoter, possibly involving NF-kappaB. CpG-oligodeoxynucleotides (CpG-ODNs), which contain unmethylated CpG dinucleotides in the context of particular base sequences, have gained attention because of their stimulating effects, via NF-kappaB, which have a strong innate immune response. To study the effects of LPS and CpG-ODNs, as well as the mechanisms of their actions regarding CMV IE enhancer/promoter activation, we used a macrophage cell line, RAW 264.7. Stimulation of the cells with LPS or CpG-ODNs resulted in the activation of the CMV IE enhancer/promoter. We examined the involvement of NF-kappaB and c-Jun transcription factors by promoter deletion/site-specific mutation analysis and ectopic expression, and found them to have additive effects. Involvement of myeloid differentiation protein, an upstream regulator of NF-kappaB and c-Jun, was also investigated. Experimental results indicate that both LPS-induced and CpG-ODN-induced activations of CMV IE enhancer/promoter are mediated by Toll-like receptor signaling molecules. Several lines of evidence suggest the potential contribution of bacterial infection in CMV reactivation along with the potential application of CpG-ODNs in gene therapy as a stimulator for the optimal expression of target genes under the control of the CMV IE enhancer/promoter.

Although MLL-AF9 caused by the chromosomal translocation t(9;11) has a critical role in acute myeloid leukemia, the molecular pathogenesis is poorly understood. Here, we identified that the cell fate determination factor DACH1 is directly up-regulated by MLL-AF9. Recently we showed that the forced expression of DACH1 in myeloid cells induced p27(Kip1) and repressed p21(Cip1), which is a pivotal characteristic of the myeloid progenitor. Consistent with our previous study, ectopic expression of DACH1 contributed to the maintenance of colonogenic activity and blocked the differentiation of myeloid progenitors. Moreover, we here identified an endogenous HOXA9-DACH1 complex mediated by the carboxyl terminus of DACH1 in t(9;11) leukemia cells. qRT-PCR revealed that DACH1 has a stronger transcription-promoting activity with HOXA9 than does PBX2 with HOXA9. Furthermore, C/EBPα and GATA-1 can directly bind to the promoter of DACH1 and act as a transcriptional suppressor. Expression of DACH1 is down-regulated during myeloid differentiation and shows an inverse pattern compared to C/EBPα and GATA-1 expression. However, ectopic expression of C/EBPα and/or GATA-1 could not abrogate the over-expression of DACH1 induced by MLL-AF9. Therefore, we postulate that the inability of C/EBPα and GATA-1 to down-regulate DACH1 expression induced by MLL-AF9 during myeloid differentiation may contribute to t(9;11) leukemogenesis.

The molecular mechanisms for epithelial differentiation have been studied by observing skin development in embryogenesis, but the early signaling modulations involved in tongue epithelial differentiation are not completely understood. Based on the gene expression patterns of the Fgf signaling molecules and previous results from Fgf10 and Fgfr2b knockout mice, it was hypothesized that there would be fundamental signaling interactions through the epithelial Fgfr2b and its mesenchymal ligand Fgf10 to regulate tongue epithelium differentiation. To elucidate these reciprocal interactions in tongue epithelial differentiation, this study employed an in vitro tongue organ culture system with antisense-oligodeoxynucleotides (AS-ODNs) and recombinant protein-soaked bead implantation for the loss-of-function and gain-of-function studies. Functional analysis of Fgf signaling revealed precise reciprocal interactions, which showed that mesenchymal Fgf10 rather than Fgf7 modulates tongue epithelial differentiation via Fgfr2b in a temporal- and spatial-specific manner.

Matrix metalloproteinase-9 (MMP-9) is a secreted type IV collagenase that plays an important role in the remodeling of the extracellular matrix (ECM) and the migration of normal and tumor cells. We have shown that CpG-ODN-induced migration of RAW 264.7 cell is regulated by MMP-9 activity by using tissue inhibitors of MMP-1 (TIMP-1). The MMP-9 gene expression was transcriptionally induced by CpG-ODN in a time-dependent manner. An MMP-9 promoter-reporter was activated by the stimulation of CpG-ODN and ectopical expression of NF-kappaB transcription factor. Inhibition of NF-kappaB nuclear localization by co-expression of a mutant IkappaBalpha protein blocked the CpG-ODN-induced MMP-9 promoter activation. BMS-345541, an IKK-2 inhibitor also inhibited the expression of MMP-9 gene induced by CpG-ODN. Direct binding of NF-kappaB protein to the promoter region of the MMP-9 was confirmed by chromatin immunoprecipitation using NF-kappaB antibody. These results lead us to a conclusion that NF-kappaB activation is required for MMP-9 gene expression. In summary, our data suggest that NF-kappaB-dependent expression of MMP-9 in response to CpG-ODN plays an important role in the recruitment of immune cells.

In rodents, the circumvallate papilla (CVP), with its underlying minor salivary gland, the von Ebners' gland (VEG), is located on the dorsal surface of the posterior tongue. Detailed morphological processes to form the proper structure of CVP and VEG have not been properly elucidated. In particular, the specific localization patterns of taste buds in CVP and the branching formation of VEG have not yet been elucidated. To understand the developmental mechanisms underlying CVP and VEG formation, detailed histological observations of CVP and VEG were examined using a three-dimensional computer-aided reconstruction method with serial histological sections and pan-Cytokeratins immunostainings. In addition, to define the developmental processes in CVP and VEG formation, we examined nerve innervations and cell proliferation using microinjections of AM1-43 and immunostainings with various markers, including phosphoinositide 3-kinase, Ki-67, PGP9.5, and Ulex europaeus agglutinin 1 (UEA1). Results revealed specific morphogenesis of CVP and VEG with nerve innervations patterns, evaluated by the coincided localization patterns of AM1-43 and UEA1. Based on these morphological and immunohistochemical results, we suggest that nerve innervations and cell proliferations play important roles in the positioning of taste buds in CVP and branching morphogenesis of VEG in tongue development.