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There exists a close connection between changes occurring in the teeth and those occurring in the jaw during the evolutionary process. In mammals, the roots of teeth are supported, along with periodontal ligaments and alveolar bones by a unique structure termed the gomphosis. In the present study, we performed combined in silico analysis using the information obtained from various DNA microarrays and identified 19 putative tooth root formation-related genes. Furthermore, quantitative PCR was performed on the candidate genes, Chd3 was confirmed as having sufficient expression levels in the early stage of tooth root formation and increased gene expression toward the middle stage. A high degree of Chd3 gene expression was observed in secretory ameloblasts and Hertwig's epithelial root sheath (HERS), but low expression was observed in developing odontoblasts and stellate reticulum. The CHD3 foci were observed in the nucleus of the HERS01a cells. In addition, knockdown experiments using SiChd3 suggested the involvement of Chd3 in the suppression of DNA synthesis. These results suggested that Chd3 plays a role in DNA synthesis in HERS cells for promoting tooth root development.

Objectives The aim of this study was to explore the relationship between the consumption of a high-fat diet and aging-dependent formation of maxillary incisor grooves in C57BL/6 mice, and to identify putative maxillary incisor groove-related genes. Methods We fed 2-month-old and 16-month-old C57BL/6 mice on either a chow diet or a high-fat diet for three months and observed changes in maxillary incisor grooves. We examined tissue sections of the maxillary incisors with grooves and carried out transcriptome analysis of the apical tissue fragments of maxillary incisors with/without grooves. Results Consumption of a high-fat diet for three months resulted in significant increases in both body weight and the number of incisor grooves. Both the number and frequency of incisor grooves increased in an age-dependent manner from 26 to 28 months, during which time an additional groove appeared. There was abnormal differentiation and apoptosis of ameloblasts on the labial surface at the grooves of the maxillary incisors. Transcriptome analysis identified 23 genes as being specific to 24-month-old mice; these included several genes related to apoptosis and cell differentiation. Conclusions The study findings indicate that, in C57BL/6 mice, consumption of a high-fat diet increases labial groove formation in maxillary incisors, which is related to aging of the tissue stem cells in the apical root end of the teeth.

High-resolution scanning electrochemical cell microscopy (SECCM) is used to image and quantitatively analyze the hydrogen evolution reaction (HER) catalytically active sites of 1H-MoS2 nanosheets, MoS2 , and WS2 heteronanosheets. Using a 20 nm radius nanopipette and hopping mode scanning, the resolution of SECCM was beyond the optical microscopy limit and visualized a small triangular MoS2 nanosheet with a side length of ca. 130 nm. The electrochemical cell provides local cyclic voltammograms with a nanoscale spatial resolution for visualizing HER active sites as electrochemical images. The HER activity difference of edge, terrace, and heterojunction of MoS2 and WS2 were revealed. The SECCM imaging directly visualized the relationship of HER activity and number of MoS2 nanosheet layers and unveiled the heterogeneous aging state of MoS2 nanosheets. SECCM can be used for improving local HER activities by producing sulfur vacancies using electrochemical reaction at the selected region.

Periodic patterning of iterative structures is diverse across the animal kingdom. Clarifying the molecular mechanisms involved in the formation of these structure helps to elucidate the process of organogenesis. Turing-type reaction-diffusion mechanisms have been shown to play a critical role in regulating periodic patterning in organogenesis. Palatal rugae are periodically patterned ridges situated on the hard palate of mammals. We have previously shown that the palatal rugae develop by a Turing-type reaction-diffusion mechanism, which is reliant upon Shh (as an inhibitor) and Fgf (as an activator) signaling for appropriate organization of these structures. The disturbance of Shh and Fgf signaling lead to disorganized palatal rugae. However, the mechanism itself is not fully understood. Here we found that Lrp4 (transmembrane protein) was expressed in a complementary pattern to Wise (a secreted BMP antagonist and Wnt modulator) expression in palatal rugae development, representing Lrp4 expression in developing rugae and Wise in the inter-rugal epithelium. Highly disorganized palatal rugae was observed in both Wise and Lrp4 mutant mice, and these mutants also showed the downregulation of Shh signaling, which was accompanied with upregulation of Fgf signaling. Wise and Lrp4 are thus likely to control palatal rugae development by regulating reaction-diffusion mechanisms through Shh and Fgf signaling. We also found that Bmp and Wnt signaling were partially involved in this mechanism.

Labial grooves in maxillary incisors have been reported in several wild-type rodent species. Previous studies have reported age-dependent labial grooves occur in moderate prevalence in C57BL/6 mice; however, very little is known about the occurrence of such grooves. In the present study, we observed age-dependent groove formation in C57BL/6 mice up to 26 months after birth and found that not only the frequency of the appearance of incisor grooves but also the number of grooves increased in an age-dependent manner. We examined the molecular mechanisms of age-dependent groove formation by performing DNA microarray analysis of the incisors of 12-month-old (12M) and 24-month-old (24M) mice. Amelx, encoding the major enamel matrix protein AMELOGENIN, was identified as a 12M-specific gene. Comparing with wild-type mice, the maxillary incisors of Amelx-/- mutants indicated the increase of the frequency and number of labial grooves. These findings suggested that the Amelx gene impacts the age-dependent appearance of the labial incisor groove in C57BL/6 mice.

Bitter taste avoidance behavior (BAB) plays a fundamental role in the avoidance of toxic substances with a bitter taste. However, the molecular basis underlying the development of BAB is unknown. To study critical developmental events by which taste buds turn into functional organs with BAB, we investigated the early phase development of BAB in postnatal mice in response to bitter-tasting compounds, such as quinine and thiamine. Postnatal mice started to exhibit BAB for thiamine and quinine at postnatal day 5 (PD5) and PD7, respectively. Histological analyses of taste buds revealed the formation of microvilli in the taste pores starting at PD5 and the localization of type 2 taste receptor 119 (TAS2R119) at the microvilli at PD6. Treatment of the tongue epithelium with cytochalasin D (CytD), which disturbs ACTIN polymerization in the microvilli, resulted in the loss of TAS2R119 localization at the microvilli and the loss of BAB for quinine and thiamine. The release of ATP from the circumvallate papillae tissue due to taste stimuli was also declined following CytD treatment. These results suggest that the localization of TAS2R119 at the microvilli of taste pores is critical for the initiation of BAB.

Tooth and bone are major tissues involved in physiological calcification in the body, and they use similar molecular pathways for development, homeostasis, and regeneration. Harmine (HMN) is a natural small compound that stimulates osteoblast differentiation in vitro and in vivo. Here we examined the biological effect of HMN on the postnatal development of molar tooth roots and periodontal tissues. HMN supported the formation of tooth roots and periodontal tissues in developing tooth germs. In tooth germ organ culture, HMN promoted the elongation of Hertwig's epithelial root sheath (HERS) and stimulated cell proliferation in HERS and dental follicle-derived tissues, including dental papillae and dental follicles. HMN stimulated cell proliferation and cell movement of HERS-derived cells without mesenchymal cells in vitro and directly induced the phosphorylation of SMAD1/5/8 protein in HERS-derived cells. Our results indicated that HMN was the first natural small compound to stimulate postnatal development of tooth germs.

Background: The brain vascular system arises from the perineural vascular plexus (PNVP) which sprouts radially into the neuroepithelium and subsequently branches off laterally to form a secondary plexus in the subventricular zone (SVZ), the subventricular vascular plexus (SVP). The process of SVP formation remains to be fully elucidated. We investigated the role of Foxc1 in early stage vascular formation in the ventral telencephalon. Results: The Foxc1 loss of function mutant mouse, Foxc1ch/ch, showed enlarged telencephalon and hemorrhaging in the ventral telencephalon by embryonic day 11.0. The mutant demonstrated blood vessel dilation and aggregation of endothelial cells in the SVZ after the invasion of endothelial cells through the radial path, which lead to failure of SVP formation. During this early stage of vascular development, Foxc1 was expressed in endothelial cells and pericytes, as well as in cranial mesenchyme surrounding the neural tube. Correspondingly, abnormal deposition pattern of basement membrane proteins around the vessels and increased strong Vegfr2 staining dots were found in the aggregation sites. Conclusions: These observations reveal an essential role for Foxc1 in the early stage of vascular formation in the telencephalon. Developmental Dynamics 244:703–711, 2015. © 2015 Wiley Periodicals, Inc.

Nuclear factor kappa B (NF-κB) signaling plays critical roles in many physiological and pathological processes, including regulating organogenesis. Down-regulation of NF-κB signaling during development results in hypohidrotic ectodermal dysplasia. The roles of NF-κB signaling in tooth development, however, are not fully understood. We examined mice overexpressing IKKβ, an essential component of the NF-κB pathway, under keratin 5 promoter ( K5-Ikkβ). K5-Ikkβ mice showed supernumerary incisors whose formation was accompanied by up-regulation of canonical Wnt signaling. Apoptosis that is normally observed in wild-type incisor epithelium was reduced in K5-Ikkβ mice. The supernumerary incisors in K5-Ikkβ mice were found to phenocopy extra incisors in mice with mutations of Wnt inhibitor, Wise. Excess NF-κB activity thus induces an ectopic odontogenesis program that is usually suppressed under physiological conditions.

Fibroblast growth factor (FGF) signaling is involved in skeletal development. Among total 22 FGFs, it is suggested that FGF18 functions in promotion of osteoblast differentiation. In order to elucidate the mechanism of FGF18-dependent acceleration of osteogenesis, we implanted rhFGF18 soaked beads over mouse fetal coronal sutures using ex-utero surgery. The coronal suture area comprises the peripheries of the developing frontal and parietal bones, separated by the sutural mesenchyme. rhFGF18 accelerated osteogenesis by promoting connection of the frontal and parietal bone domains, resulting in elimination of the sutural mesenchyme. Expression of Fgf receptors, Fgfr1, -2 and -3 involved in skeletal development, was maintained or upregulated in the developing bone domains, consistent with enhanced osteogenesis. Bone morphogenetic protein (Bmp) 2 was specifically upregulated in the skeletogenic layer and the application of Bmp antagonist, rmNoggin, inhibited rhFGF18-dependent upregulation of osteoblast markers. These results suggest that FGF18 accelerates osteogenesis by upregulation of Bmp2 as well as maintenance or upregulation of Fgfr1, -2 and -3 expression in osteoblasts.

Hertwig's epithelial root sheath (HERS), a bilayered epithelial cell sheath located at the cervical loop of the enamel organ in a developing tooth, is at the forefront of root formation. However, little is known about the exact mechanisms that regulate the development of HERS. The neuropeptide vasoactive intestinal peptide (VIP) is involved in the development of various tissues and cells. In this study, we investigated the roles of VIP in HERS development. VIP-immunoreactive nerve fibers were found in the dental pulp and around the root apex of the tooth, while the expression of VIP receptor 1 (VPAC1) was observed in HERS. The expression level of VPAC1 correlated with the development of HERS and was elevated at postnatal days 14 and 21. Using ex vivo cultures of neonatal tooth germs, VIP enhanced the elongation and proliferation of HERS. In vitro, VIP also promoted the proliferation of cells from the HERS-derived cell line, HERS01a cells, and upregulated the mRNA expression of cytokeratin 14 and vimentin (typical molecular markers of HERS) in these cells. These results suggest that VIP may be an essential factor for HERS development.

Date Y, Yokoyama Y, Kondo H, Kuroda S, Ohya K, Ota MS, Iseki S, Kasugai S. Restricted expression of chromatin remodeling associated factor Chd3 during tooth root development. J Periodont Res 2012; 47: 180–187. © 2011 John Wiley & Sons A/S Background and Objective: The tooth root is one of the critical parts to maintain tooth function; however, the molecular mechanisms of root development remain unknown. We aimed to identify specific factors for root morphogenesis using a newly developed experimental system. Material and Methods: Tentative cementoblasts and periodontal ligament cells from mouse mandibular molars were isolated using laser capture microdissection. More than 500 cementoblasts and periodontal ligament cells were separately captured. After RNA extraction and amplification, mRNA expression in isolated cementoblasts was compared with that of periodontal ligament cells by cDNA microarray analysis. Then, putative cementoblast-specific genes were subjected to in situ hybridization analysis to confirm the results in mouse mandible. Results: Approximately 2000 genes were differentially expressed between these tissues. Among those genes, zinc finger helicase (ZFH), also termed chromodomain-helicase-DNA-binding protein 3 (Chd3), was one of the highly expressed transcripts in tentative cementoblasts. In situ hybridization revealed that ZFH/Chd3 was strongly expressed in Hertwig’s epithelial root sheath rather than in cementum. Moreover, its expression disappeared when root formation was advanced in the first molar. In contrast, Chd3 was continuously expressed in dental epithelial cells of the cervical loop, in which root extension is never terminated. Conclusion: These results suggest that ZFH/Chd3 might play an important role in tooth root development and subsequent cementogenesis.

Mammalian dentitions consist of different shapes/types of teeth that are positioned in different regions of the jaw (heterodont) whereas in many fish and reptiles all teeth are of similar type (homodont). The process by which heterodont dentitions have evolved in mammals is not understood. In many teleosts teeth develop in the pharynx from endoderm (endodermal teeth), whereas mammalian teeth develop from the oral ectoderm indicating that teeth can develop (and thus possibly evolve) via different mechanisms. In this article, we compare the molecular characteristics of pharyngeal/foregut endoderm with the molecular characteristics of oral ectoderm during mouse development. The expression domains of Claudin6, Hnf3beta, alpha-fetoprotein, Rbm35a, and Sox2 in the embryonic endoderm have boundaries overlapping the molar tooth-forming region, but not the incisor region in the oral ectoderm. These results suggest that molar teeth (but not incisors) develop from epithelium that shares molecular characteristics with pharyngeal endoderm. This opens the possibility that the two different theories proposed for the evolution of teeth may both be correct. Multicuspid (eg. molars) having evolved from the externalization of endodermal teeth into the oral cavity and monocuspid (eg. incisors) having evolved from internalization of ectodermal armour odontodes of ancient fishes. The two different mechanisms of tooth development may have provided the developmental and genetic diversity on which evolution has acted to produce heterodont dentitions in mammals.

Previously, the effects of several bitter substances have been investigated in the contraction of the murine jejunum and ileum, reporting that these independently suppress the rhythm generation of the interstitial cells of Cajal. Recently, it was reported that thiamin, which binds to a bitter taste receptor, modifies the early phase of the ileum contraction, whereas the physiological effects on the rhythm and amplitude of jejunum and ileum contractions remain unclear. In this study, it was investigated the physiological effects of thiamin and quinine on the in vitro contraction of the murine jejunum and ileum using mice for all experiments. the periodic contraction of the jejunum was observed before the administration of acetylcholine (Ach) and other substances, and the tonic amplitudes induced by the substances. These bitter substances variably affect the Ach-induced rhythmic contraction of the jejunum and ileum in vitro. In addition, quinine hydrochloride (Qui) and thiamin hydrochloride (Thi) variably affect the Ach-induced rhythmic contraction of the jejunum and ileum in vitro. Both Qui and Thi markedly increase the rhythmic contraction in the jejunum. Although Thi does not change the rhythmic contraction in the ileum, it gradually reduces the amplitude in the jejunum. Conversely, Qui gradually reduces the amplitude and almost inhibits the contraction in the jejunum. Furthermore, an antagonist of the adrenalin-beta3 receptor, SR59230A, enhances the Qui-induced inhibition of the contraction in the jejunum.

Thiamine and quinine are popular bitter substances and their physiological effects have been studied; however, their impact on digestion remains unknown. Here, the physiological effects of thiamine and quinine was investigated for in vitro contraction of mouse ileum. Acetylcholine stimulates autonomous contraction of mouse ileum in a dose-dependent manner. The effect of Acetylcholine for contraction of ileum was partly suppressed by the adrenaline administration. Upon simultaneous treatment of the ileum by acetylcholine, thiamine, and quinine decreased the maximum contraction. The period till half maximum contraction was prolonged by the presence of thiamine and quinine but not by adrenaline. Because a physiological effect of thiamine and quinine was observed on acetylcholine-induced contraction of the ileum, the repertoire of human bitter taste receptors, TAS2R-1, -4, -7, -10, -14, -31, -39, -40, -43, and -46, were investigated to which thiamine and quinine may bind. These human bitter taste receptors were further analyzed among the database for mouse homologs using evolutionally conserved amino acid sequences. The only bitter receptor for both thiamine and quinine was TAS2R-39, the homology of TAS2R-139 to human TAS2R-39 was 74%. Importantly, the homology of mouse TAS2R-119 to human TAS2R-1 which interact with thiamine was 91%, and that of TAS2R-130 to human TAS2R-7 that interact with quinine was 81%. The present study indicated that thiamine and quinine changed the early phase of contraction of ileum in mice and suggested that TAS2R119 and TAS2R130 expressed in mouse enteroendocrine cells to modify the physiological effects of thiamine and quinine on the acetylcholine-induced contraction of the ileum.

Changes in tooth shape have played a major role in vertebrate evolution with modification of dentition allowing an organism to adapt to new feeding strategies. The current view is that molar teeth evolved from simple conical teeth, similar to canines, by progressive addition of extra “cones” to form progressively complex multicuspid crowns. Mammalian incisors, however, are neither conical nor multicuspid, and their evolution is unclear. We show that hypomorphic mutation of a cell surface receptor, Lrp4, which modulates multiple signaling pathways, produces incisors with grooved enamel surfaces that exhibit the same molecular characteristics as the tips of molar cusps. Mice with a null mutation ofLrp4develop extra cusps on molars and have incisors that exhibit clear molar-like cusp and root morphologies. Molecular analysis identifies misregulation of Shh and Bmp signaling in the mutant incisors and suggests an uncoupling of the processes of tooth shape determination and morphogenesis. Incisors thus possess a developmentally suppressed, cuspid crown-like morphogenesis program similar to that in molars that is revealed by loss of Lrp4 activity. Several mammalian species naturally possess multicuspid incisors, suggesting that mammals have the capacity to form multicuspid teeth regardless of location in the oral jaw. Localized loss of enamel may thus have been an intermediary step in the evolution of cusps, both of which useLrp4-mediated signaling.

Tooth morphogenesis is regulated by reciprocal interactions between the dental epithelium and odontogenic mesenchyme. As tooth roots are fundamental structures of the tooth support system, the morphology and functions of the roots are very important. However, basic information on the morphology of tooth root patterning and the molecular mechanism of root morphogenesis is largely unavailable. Following tooth crown formation, the dental epithelium forms a double-layered Hertwig's epithelial root sheath (HERS) derived from inner and outer enamel epithelium. Previous studies have reported that HERS plays an important role in tooth root development. Here, we report the correlation between the number of major cusps of the tooth crown and number of tooth roots of first molar and last premolar teeth in several extant mammals. We also discuss the molecular mechanism of tooth root patterning by introducing studies of mouse mutants and human syndromes associated with an abnormal molar morphology.

Diet is one of the most important factors driving the process of natural selection in evolution. The dentition is adapted for the diet, contributing to the diversification and adaptation of extant vertebrates. Importantly, the structure of the molar shows marked variation in extant mammals to facilitate the processing of many kinds of food and support the occlusal force effectively. A tooth root is an important element, connecting a tooth with the jaw to support the occlusal force. However, morphological studies on teeth mainly focus on the crown, whereas there has been an insufficient accumulation of information on the morphogenic control mechanism of tooth root structures. There have been some reports on the root form in humans due to clinical demand, and recent studies on Hertwig's epithelial root sheath (HERS), which plays an important role in root development, have progressed rapidly. However, studies on the regulatory mechanism of the tooth root structure in relation to the jaw and crown are still awaited. In this paper, we highlight the following three issues : the morphological relevance of the tooth root and crown, and developmental mechanisms and phylogenetic aspects of the tooth roots. HERS produces several signaling molecules including bone morphogenic protein (BMP), Sonic Hedgehog (Shh), Wnt, ectodysplasin (Eda), and fibrobast growth factor (FGF). These factors regulate the closure of the pulp chamber floor to determine the numbers and width of the tooth roots, and then regulate the fusion, curve, and length. The tooth root differentiates and develops in conjunction with its crown. However, few studies have examined the tooth root from an evolutionary perspective. We need to advance research on the tooth root including the whole tooth in developmental biology, evolutionary biology, and comparative odontology.

Cell-based therapy combined with tissue stem cells and tissue-engineering technology is believed to be a very powerful tool for regeneration of the tooth root and periodontal tissue in the future. The molecular mechanism of tooth root development is useful for this therapy ; however, not enough basic information has been accumulated about tooth root development. Reciprocal epithelial-mesenchymal interactions, polarized growth and complicated morphogenic events are involved in tooth development. Recently, a Sonic hedgehog (Shh) -fibroblast growth factor (Fgfs) -dependent regulatory mechanism was found in the developing tooth roots, in a previous report that dental tissues had zone polarizing activity (ZPA) in the chick limb bud. Shh-Fgfs morphogenetic signaling is conserved in the developing molar tooth roots, but a unique combination of Fgfs is used for molar tooth root development compared to the limb bud patterning process.

Tissue engineering approaches have been developed for vascular grafts, but success has been limited to arterial replacements of large-caliber vessels. We have developed an innovative technique to transplant engineered capillary networks by printing techniques. Endothelial cells were cultured on a patterned substrate, in which network patterns were generated by prior optical lithography. Subsequently, the patterned cells were transferred to extracellular matrix and tissue at which point they changed their morphologies and formed tubular structures. Microinjection of dye showed that the micrometer-scale tubular structure had in vitro flow potential. When capillary-like networks engineered on amnion membranes were transplanted into mice, we found blood cells inside of the lumen of the transplanted capillary-like structure. This is the first report of the in vitro formation of capillary networks using cell transfer technique, and this novel technique may open the way for development of rapid and effective blood perfusion systems in regenerative medicine.

Autotaxin (ATX) is a cancer-associated motogen that has multiple biological activities in vitro through the production of bioactive small lipids, lysophosphatidic acid (LPA). ATX and LPA are abundantly present in circulating blood. However, their roles in circulation remain to be solved. To uncover the physiological role of ATX we analyzed ATX knock-out mice. In ATX-null embryos, early blood vessels appeared to form properly, but they failed to develop into mature vessels. As a result ATX-null mice are lethal around embryonic day 10.5. The phenotype is much more severe than those of LPA receptor knock-out mice reported so far. In cultured allantois explants, neither ATX nor LPA was angiogenic. However, both of them helped to maintain preformed vessels by preventing disassembly of the vessels that was not antagonized by Ki16425, an LPA receptor antagonist. In serum from heterozygous mice both lysophospholipase D activity and LPA level were about half of those from wild-type mice, showing that ATX is responsible for the bulk of LPA production in serum. The present study revealed a previously unassigned role of ATX in stabilizing vessels through novel LPA signaling pathways.

Cajal bodies contain cyclin E/cdk2 and the substrate p220 NPAT to regulate the transcription of histones, which is essential for cell proliferation, however, recent mouse knockout studies indicate that cyclin E and cdk2 are dispensable for these events. Because the CBP/p300 histone acetyltransferase are also known to be involved in cell proliferation, we examined the molecular and functional interactions of p220 NPAT with the CBP/p300 at the G1/S boundary as cell cycle regulators. The subnuclear localization of p220 NPAT and CBP/p300 proteins showed that their foci partially overlapped in a cell cycle dependent manner. Overexpression of p220 NPAT and CBP/p300 cooperatively enhanced G1/S transition and DNA synthesis even without cdk2 phosphorylation site. Finally, molecular alignment analysis indicated that p220 NPAT contains several potential substrate sites for CBP/p300. Overall, our findings demonstrate that p220 NPAT and CBP/p300 form a transient complex at the G1/S boundary to play cooperative roles to promote the S-phase entry.

The brain vascular system arises from the perineural vascular plexus (PNVP) which sprouts radially into the neuroepithelium and subsequently branches off laterally to form a secondary plexus in the subventricular zone (SVZ), the subventricular vascular plexus (SVP). The process of SVP formation remains to be fully elucidated. We investigated the role of Foxc1 in early stage vascular formation in the ventral telencephalon.The Foxc1 loss of function mutant mouse, Foxc1(ch/ch) , showed enlarged telencephalon and hemorrhaging in the ventral telencephalon by embryonic day 11.0. The mutant demonstrated blood vessel dilation and aggregation of endothelial cells in the SVZ after the invasion of endothelial cells through the radial path, which lead to failure of SVP formation. During this early stage of vascular development, Foxc1 was expressed in endothelial cells and pericytes, as well as in cranial mesenchyme surrounding the neural tube. Correspondingly, abnormal deposition pattern of basement membrane proteins around the vessels and increased strong Vegfr2 staining dots were found in the aggregation sites.These observations reveal an essential role for Foxc1 in the early stage of vascular formation in the telencephalon.

Stress is a well-known cause of numerous digestive conditions, including gastrointestinal-function disorders. The autonomic nervous system regulates intestinal movements via cholinergic and adrenergic efferent fibers; however it is not clear how stress could affect these control mechanisms and in particular whether in a site-dependent manner. In this study we tested in vitro the effects of topical application of acetylcholine (Ach) and adrenalin (Adr) on smooth-muscle contractions of intestinal segments isolated from stress-conditioned rats. Stress was loaded by hypergravity stimulation (10 min/day) for periods of 1, 6 or 30 days. As a result, stress-conditioning affected intestinal sensitivity to Ach and Adr differently at sections of the ileum and colon. In the ileum no significant differences were found between control and stress-conditioned rats, whereas in the colon, samples from 6- and 30-day stress-conditioned rats showed larger amplitudes of Ach-induced contraction, as well as greater antagonization by Adr application. These results suggest that stress conditioning can modify autonomic control of intestinal movements by altering smooth-muscle sensitivity to Ach and Adr.

Fibroblast growth factors (FGFs) regulate the proliferation and differentiation of various cells via their respective receptors (FGFRs). During the early stages of tooth development in fetal mice, FGFs and FGFRs have been shown to be expressed in dental epithelia and mesenchymal cells at the initial stages of odontogenesis and to regulate cell proliferation and differentiation. However, little is known about the expression patterns of FGFs in the advanced stages of tooth development. In the present study, we focused on FGF18 expression in the rat mandibular first molar (M1) during the postnatal crown and root formation stages. FGF18 signals by RT-PCR using cDNAs from M1 were very weak at postnatal day 5 and were significantly up-regulated at days 7, 9 and 15. Transcripts were undetectable by in situ hybridization (ISH) but could be detected by in situ RT-PCR in the differentiated odontoblasts and cells of the sub-odontoblastic layer in both crown and root portions of M1 at day 15. The transcripts of FGFR2c and FGFR3, possible candidate receptors of FGF18, were detected by RT-PCR and ISH in differentiated odontoblasts throughout postnatal development. These results suggest the continual involvement of FGF18 signaling in the regulation of odontoblasts during root formation where it may contribute to dentin matrix formation and/or mineralization.

Fibroblast growth factor (FGF) signaling is involved in skeletal development. Among total 22 FGFs, it is suggested that FGF18 functions in promotion of osteoblast differentiation. In order to elucidate the mechanism of FGF18-dependent acceleration of osteogenesis, we implanted rhFGF18 soaked beads over mouse fetal coronal sutures using ex-utero surgery. The coronal suture area comprises the peripheries of the developing frontal and parietal bones, separated by the sutural mesenchyme. rhFGF18 accelerated osteogenesis by promoting connection of the frontal and parietal bone domains, resulting in elimination of the sutural mesenchyme. Expression of Fgf receptors, Fgfr1, -2 and -3 involved in skeletal development, was maintained or upregulated in the developing bone domains, consistent with enhanced osteogenesis. Bone morphogenetic protein (Bmp) 2 was specifically upregulated in the skeletogenic layer and the application of Bmp antagonist, rmNoggin, inhibited rhFGF18-dependent upregulation of osteoblast markers. These results suggest that FGF18 accelerates osteogenesis by upregulation of Bmp2 as well as maintenance or upregulation of Fgfr1, -2 and -3 expression in osteoblasts.

Hertwig's epithelial root sheath (HERS), a bilayered epithelial cell sheath located at the cervical loop of the enamel organ in a developing tooth, is at the forefront of root formation. However, little is known about the exact mechanisms that regulate the development of HERS. The neuropeptide vasoactive intestinal peptide (VIP) is involved in the development of various tissues and cells. In this study, we investigated the roles of VIP in HERS development. VIP-immunoreactive nerve fibers were found in the dental pulp and around the root apex of the tooth, while the expression of VIP receptor 1 (VPAC1) was observed in HERS. The expression level of VPAC1 correlated with the development of HERS and was elevated at postnatal days 14 and 21. Using ex vivo cultures of neonatal tooth germs, VIP enhanced the elongation and proliferation of HERS. In vitro, VIP also promoted the proliferation of cells from the HERS-derived cell line, HERS01a cells, and upregulated the mRNA expression of cytokeratin 14 and vimentin (typical molecular markers of HERS) in these cells. These results suggest that VIP may be an essential factor for HERS development.

To create a drug delivery system that allows the controlled release of proteins, such as growth factors, over a long-term period, cholesteryl group- and acryloyl group-bearing pullulan (CHPOA) nanogels were aggregated to form fast-degradable hydrogels (CHPOA/hydrogels) by cross-linking with thiol-bearing polyethylene glycol. The gold standard of clinical bone reconstruction therapy with a physiologically active material is treatment with recombinant human bone morphogenetic protein 2 (BMP2); however, this approach has limitations, such as inflammation, poor cost-efficiency, and varying interindividual susceptibility. In this study, two distinct growth factors, BMP2 and recombinant human fibroblast growth factor 18 (FGF18), were applied to a critical-size skull bone defect for bone repair by the CHPOA/hydrogel system. The CHPOA-FGF18/hydrogel displayed identical results to the control CHPOA-PBS/hydrogel, and the CHPOA-BMP2/hydrogel treatment imperfectly induced bone repair. By contrast, the CHPOA-FGF18 + BMP2/hydrogel treatment strongly enhanced and stabilized the BMP2-dependent bone repair, inducing osteoprogenitor cell infiltration inside and around the hydrogel. This report indicates that the CHPOA/hydrogel system can successfully deliver two different proteins to the bone defect to induce effective bone repair. The combination of the CHPOA/hydrogel system with the growth factors FGF18 and BMP2 might be a step towards efficient bone tissue engineering.

The low-density lipoprotein (LDL) receptor family is a large evolutionarily conserved group of transmembrane proteins. It has been shown that LDL receptor family members can also function as direct signal transducers or modulators for a broad range of cellular signaling pathways. We have identified a novel mode of signaling pathway integration/coordination that occurs outside cells during development that involves an LDL receptor family member. Physical interaction between an extracellular protein (Wise) that binds BMP ligands and an Lrp receptor (Lrp4) that modulates Wnt signaling, acts to link these two pathways. Mutations in either Wise or Lrp4 in mice produce multiple, but identical abnormalities in tooth development that are linked to alterations in BMP and Wnt signaling. Teeth, in common with many other organs, develop by a series of epithelial-mesenchymal interactions, orchestrated by multiple cell signaling pathways. In tooth development, Lrp4 is expressed exclusively in epithelial cells and Wise mainly in mesenchymal cells. Our hypothesis, based on the mutant phenotypes, cell signaling activity changes and biochemical interactions between Wise and Lrp4 proteins, is that Wise and Lrp4 together act as an extracellular mechanism of coordinating BMP and Wnt signaling activities in epithelial-mesenchymal cell communication during development.

Tenascin-W is a tenascin family member that forms part of a complex extracellular matrix, and previous studies have suggested its association with osteogenesis. In the present study we investigated the roles of tenascin-W in osteogenesis. We found that tenascin-W is expressed in osteoblasts at the edge of the developing bone domain prior to mineralization in mouse fetuses. Expression of tenascin-W was induced during the course of mineralization of the Kusa-A1 osteoblast cell line. In the interfrontal suture of postnatal mice, the anterior portion remains patent and the posterior portion closes by 4 weeks of age. Tenascin-W expression was downregulated at 1 week of age in the posterior frontal suture, whereas in the anterior suture, expression was maintained until the mice reached 4 weeks of age. Fibroblast growth factor 2 (FGF2)-bead application to the mouse fetal skull by ex-utero surgery accelerated osteoblast differentiation, but inhibited mineralization with a downregulation of tenascin-W expression. These results suggest that tenascin-W is involved in osteoblast maturation (i.e. mineralization).

Avoiding the limitations of the adult liver niche, transplantation of hepatic stem/progenitor cells into fetal liver is desirable to analyze immature cells in a hepatic developmental environment. Here, we established a new monitor tool for cell fate of hepatic progenitor cells transplanted into the mouse fetal liver by using ex utero surgery. When embryonic day (ED) 14.5 hepatoblasts were injected into the ED14.5 fetal liver, the transplanted cells expressed albumin abundantly or alpha-fetoprotein weakly, and contained glycogen in the neonatal liver, indicating that transplanted hepatoblasts can proliferate and differentiate in concord with surrounding recipient parenchymal cells. The transplanted cells became mature in the liver of 6-week-old mice. Furthermore, this method was applicable to transplantation of hepatoblast-like cells derived from mouse embryonic stem cells. These data indicate that this unique technique will provide a new in vivo experimental system for studying cell fate of hepatic stem/progenitor cells and liver organogenesis.

The fourth author's name appears incorrectly. It should be: Hong Y. Choi. The correct citation should read: Ohazama A, Johnson EB, Ota MS, Choi HY, Porntaveetus T, et al. (2008) Lrp4 Modulates Extracellular Integration of Cell Signaling Pathways in Development. PLoS ONE 3(12): e4092. doi:10.1371/journal.pone.0004092

Sansho (Japanese pepper) is a common spice widely used in Japanese cuisine. In addition, it is also an important component in Kampo medicine, such as in Daiken Chuto, which stimulates gastrointestinal motility and improves postoperative ileus. On the other hand, we previously reported that gravity stress loading produce negative effects on ileal movements. In the present study, we report changes in ileal motility after gravity-stress with and without Sansho intake in male and female rats. Ileal movements were activated by topical Acetylcholine (Ach) application, and maximum amplitudes (MA) of the evoked contraction were compared. Clear tonic patterns were observed in the ileal motility after Ach application. After short-term stress, there were no significant differences in MA between control and Sansho-intake groups in both sexes. However, after long-term stress-loading, significant enhancement of MA was observed in Sansho-intake group in males, but not in females. The present study showed that SAN affected Ach-induced ileal motility in the male, but not in the female after long-term stress loading, indicating sex differences in effects of Sansho intake. It is suggested that Sansho is more effective in males than in females to decrease negative stress response.

We investigated the effects of somatostatin on the ileal movements after gravity stress and compared these effects between male and female rats. Using an in vitro preparation, measurements of ileal movements evoked by somatostatin application were done at 1, 3, 15 and 30 days after 3G gravity loading-conditioning. Mixed phasic and tonic patterns were observed in the ileal muscle activities. Gravity-stress decreased or antagonized somatostatin inhibitory effects on tonic ileal contraction at an early stage (at day 3) in females, but not in males, indicating that sex differences exist in the way that changes of somatostatin sensitivity is modulated by stress conditioning.

Ginger is widely used as a spice and also an effective herbal medicine to treat gastrointestinal disorders. On the other hand, stress can induce various modulation of digestive motility. Here, we investigated ginger effects on stress-induced motility of the isolated ileum in male and female rats, in vitro. Rats (Wistar, SPF, 7-12 weeks of age, 148-393 g BW) were divided into Control (1G) and 3G groups. 3G stress (every day for 10 min) was loaded by centrifugal apparatus for 1, 3, 15 and 30 days. After the stress loading at each day, a 1 cm-long section of the ileum was isolated under barbiturate anesthesia and fixed to a Magnus-type chamber filled with Tyrode solution. Ileal movements were observed for 60 s following application of zingerone, which is also called vanillylacetone and a key component of the pungency of ginger (0.1-10 μM). Spontaneous motility movements with phasic and tonic patterns were observed in the ileum. The former was a peristalsis-like movement and the latter was a slow fluctuation of the baseline. Ginger induced enhanced effects on the rhythmic phasic motility in relation to amplitude. In the female, 3G gravity loading had no effects on the ginger-induced motility, however, suppressive effect of the ginger-induced phasic movements was clearly observed at day 15 in the male. The present study showed that gravity stress changed ginger-induced effects on phasic ileal motility in the male, but not in the female, indicating that sex differences were observed in the ginger effects modulated by stress loading.

Hertwig’s epithelial root sheath (HERS) is important for tooth root formation, but the molecular basis for the signaling of root development remains uncertain. We hypothesized that Sonic hedgehog (Shh) signaling is involved in the HERS function, because it mediates epithelial-mesenchymal interactions during embryonic odontogenesis. We examined the gene expression patterns of Shh signaling in murine developing molar roots. Shh and Patched2 transcripts were identified in the HERS, whereas Patched1, Smoothened, and Gli1 were expressed in the proliferative dental mesenchyme in addition to the HERS. To confirm whether Shh signaling physiologically functions in vivo, we analyzed mesenchymal dysplasia ( mes) mice carrying an abnormal C-terminus of the PATCHED1 protein. In the mutant, cell proliferation was repressed around the HERS at 1 wk. Moreover, the molar eruption was disturbed, and all roots were shorter than those in control littermates at 4 wks. These results indicate that Shh signaling is important in tooth root development. Abbreviations used: BrdU, 5-bromo-2′-deoxyuridine; HERS, Hertwig’s epithelial root sheath; NFI-C/CTF, nuclear factor Ic/CAAT box transcription factor; PCNA, proliferating cell nuclear antigen; Ptc, patched; Shh, sonic hedgehog; Smo, smoothened.

The sense of taste is of critical importance to animal survival. Although studies of taste signal transduction mechanisms have provided detailed information regarding taste receptor calcium signaling molecules (TRCSMs, required for sweet/bitter/umami taste signal transduction), the ontogeny of taste cells is still largely unknown. We used a novel approach to investigate the molecular regulation of taste system development in mice by combining in silico and in vivo analyses. After discovering that TRCSMs colocalized within developing circumvallate papillae (CVP), we used computational analysis of the upstream regulatory regions of TRCSMs to investigate the possibility of a common regulatory network for TRCSM transcription. Based on this analysis, we identified Hes1 as a likely common regulatory factor, and examined its function in vivo. Expression profile analyses revealed that decreased expression of nuclear HES1 correlated with expression of type II taste cell markers. After stage E18, the CVP of Hes1−/ − mutants displayed over 5-fold more TRCSM-immunoreactive cells than did the CVP of their wild-type littermates. Thus, according to our composite analyses, Hes1 is likely to play a role in orchestrating taste cell differentiation in developing taste buds., Author Summary The sensation of taste is composed of five basic modalities: sweet, bitter, umami, sour, and salty. Specialized taste cells perceive the various chemical cues within food. About 100 taste cells assemble into onion-shaped clusters called taste buds, which are located on taste papillae in the tongue epithelium and on oral mucosa. Of the five taste modalities, the taste stimulants responsible for sweet, bitter, and umami tastes are recognized by a group of G protein–coupled taste receptors, and the signal transduction pathways utilized following receptor stimulation share common molecules. However, it is still largely unknown how these molecules are regulated during taste cell development. We performed computer analyses based on previously known information about signal transduction pathways involved in the taste-sensing system to identify taste stem cells/progenitor factors of type II taste cells (responsible for sweet, bitter, and umami taste sensations). We found several transcription factors likely to bind to the regulatory regions of taste-related calcium signaling molecules (TRCSMs), and identified Hes1 as a potential candidate for common regulatory factors of TRCSMs. In vivo analyses using wild-type and Hes1 mutant mice confirmed that Hes1 regulates differentiation of bitter-, sweet-, and umami-sensing cells.

The extent to which cell signaling is integrated outside the cell is not currently appreciated. We show that a member of the low-density receptor-related protein family, Lrp4 modulates and integrates Bmp and canonical Wnt signalling during tooth morphogenesis by binding the secreted Bmp antagonist protein Wise. Mouse mutants of Lrp4 and Wise exhibit identical tooth phenotypes that include supernumerary incisors and molars, and fused molars. We propose that the Lrp4/Wise interaction acts as an extracellular integrator of epithelial-mesenchymal cell signaling. Wise, secreted from mesenchyme cells binds to BMP's and also to Lrp4 that is expressed on epithelial cells. This binding then results in the modulation of Wnt activity in the epithelial cells. Thus in this context Wise acts as an extracellular signaling molecule linking two signaling pathways. We further show that a downstream mediator of this integration is the Shh signaling pathway.