Enam expression is regulated by Msx2.

BACKGROUND: The precise formation of mineralized dental tissues such as enamel and/or dentin require tight transcriptional control of the secretion of matrix proteins. Here, we have investigated the transcriptional regulation of the second most prominent enamel matrix protein, enamelin, and its regulation through the major odontogenic transcription factor, MSX2. RESULTS: Using in vitro and in vivo approaches, we identified that (a) Enam expression is reduced in the Msx2 mouse mutant pre-secretory and secretory ameloblasts, (b) Enam is an early response gene whose expression is under the control of Msx2, (c) Msx2 binds to Enam promoter in vitro, suggesting that enam is a direct target for Msx2 and that (d) Msx2 alone represses Enam gene expression. CONCLUSIONS: Collectively, these results illustrate that Enam gene expression is controlled by Msx2 in a spatio-temporal manner. They also suggest that Msx2 may interact with other transcription factors to control spatial and temporal expression of Enam and hence amelogenesis and enamel biomineralization.

Pax9's Interaction With the Ectodysplasin Signaling Pathway During the Patterning of Dentition.

In these studies, we explored for the first time the molecular relationship between the paired-domain-containing transcription factor, Pax9, and the ectodysplasin (Eda) signaling pathway during mouse incisor formation. Mice that were deficient in both Pax9 and Eda were generated, and the status of dentition analyzed in all progeny using gross evaluation and histomorphometric means. When compared to wildtype controls, Pax9+/-Eda-/- mice lack mandibular incisors. Interestingly, Fgf and Shh signaling are down-regulated while Bmp4 and Lef1 appear unaffected. These findings suggest that Pax9-dependent signaling involves the Eda pathway and that this genetic relationship is important for mandibular incisor development. Studies of records of humans affected by mutations in PAX9 lead to the congenital absence of posterior dentition but interestingly involve agenesis of mandibular central incisors. The latter phenotype is exhibited by individuals with EDA or EDAR mutations. Thus, it is likely that PAX9, in addition to playing a role in the formation of more complex dentition, is also involved with EDA signaling in the initiation of odontogenesis within the incisal domain.

An Msx2-Sp6-Follistatin Pathway Operates During Late Stages of Tooth Development to Control Amelogenesis.

BACKGROUND: Ameloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both Msx2 and Sp6 transcription factors play important role. Msx2 and Sp6 mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis. METHODS: Two different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of Msx2 or Sp6 or both. Msx2 mutant mouse embryos or pups were used for in vivo studies. In situ hybridization, semi-quantitative and quantitative real time PCR were employed to study gene expression pattern. MatInspector was used to identify several potential putative Msx2 binding sites upstream of the murine Sp6 promoter region. Chromatin Immunoprecipitation (chIP) was used to confirm the binding of Msx2 to Sp6 promoter at the putative sites. RESULTS: Using the above methods we identified that (i) Msx2 and Sp6 exhibit overlapping expression in secretory ameloblasts, (ii) Sp6 expression is reduced in the Msx2 mouse mutant secretoty ameloblasts, and (iii) that Msx2, like Sp6 inhibits follistatin expression. Specifically, our loss-of function studies by silencing Msx2 and/or Sp6 in mouse dental epithelial (LS8) cells showed significant downregulation of Sp6 but upregulation of Fst expression. Transient transfection of Msx2 overexpression plasmid, up-regulated Sp6 and downregulated Fst expression. Additionally, using MatInspector, we identified several potential putative Msx2 binding sites, 3.5 kb upstream of the murine Sp6 promoter region. By chIP, we confirmed the binding of Msx2 to Sp6 promoter at these sites, thus suggesting that Sp6 is a direct target of Msx2. CONCLUSION: Collectively, these results show that Sp6 and Msx2 work in a concerted manner to form part of a network of transcription factors that operate during later stages of tooth development controlling ameloblast life cycle and amelogenesis.

Potassium channels as potential drug targets for limb wound repair and regeneration.

BACKGROUND: Ion channels are a large family of transmembrane proteins, accessible by soluble membrane-impermeable molecules, and thus are targets for development of therapeutic drugs. Ion channels are the second most common target for existing drugs, after G protein-coupled receptors, and are expected to make a big impact on precision medicine in many different diseases including wound repair and regeneration. Research has shown that endogenous bioelectric signaling mediated by ion channels is critical in non-mammalian limb regeneration. However, the role of ion channels in regeneration of limbs in mammalian systems is not yet defined. METHODS: To explore the role of potassium channels in limb wound repair and regeneration, the hindlimbs of mouse embryos were amputated at E12.5 when the wound is expected to regenerate and E15.5 when the wound is not expected to regenerate, and gene expression of potassium channels was studied. RESULTS: Most of the potassium channels were downregulated, except for the potassium channel kcnj8 (Kir6.1) which was upregulated in E12.5 embryos after amputation. CONCLUSION: This study provides a new mouse limb regeneration model and demonstrates that potassium channels are potential drug targets for limb wound healing and regeneration.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers.

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

Skin regeneration with all accessory organs following ablation with irreversible electroporation.

Skin scar formation is a complex process that results in the formation of dense extracellular matrix (ECM) without normal skin appendages such as hair and glands. The absence of a scarless healing model in adult mammals prevents the development of successful therapies. We show that irreversible electroporation of skin drives its regeneration with all accessory organs in normal adult rats. Pulsed electric fields at 500 V, with 70 µs pulse duration and 1000 pulses delivered at 3 Hz, applied through two electrodes separated by 2 mm lead to massive cell death. However, the ECM architecture of the skin was preserved. Six months after the ablation, the epidermis, sebaceous glands, panniculus carnosus, hair follicles, microvasculature and arrector pili muscle were altogether re-formed in the entire ablated area. These results suggest a key role of the ECM architecture in the differentiation, migration and signalling of cells during scarless wound healing. Copyright © 2016 John Wiley & Sons, Ltd.

Kcnh2 and Kcnj8 interactively regulate skin wound healing and regeneration.

Previous studies indicate that ion channels are mediators of bioelectricity promoting wound closure/regeneration in nonmammalian, lower vertebrate systems. The role of ion channels however in regeneration of wounds in mammalian systems that do not regenerate as adults is not yet defined. Using a mammalian model system that allows us to determine differentially expressed genes when skin regenerates and when skin does not regenerate after wound induction, we identified two potassium channels, kcnh2 and kcnj8, to be (1) differentially expressed between the two states and (2) highly expressed after wound induction at the nonregenerative state. We also found that kcnh2 small molecule inhibitor enhanced wound healing while kcnj8 small molecule inhibitor did not. In contrast, kcnj8 activator accelerated wound healing and even augmented the effect of kcnh2 inhibition. These results provide evidence for the first time that potassium channels may mediate skin wound healing and regeneration interactively.

p38α MAPK is required for tooth morphogenesis and enamel secretion.

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678-27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α(K14) mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and ß4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.

A network of transcription factors operates during early tooth morphogenesis.

Improving the knowledge of disease-causing genes is a unique challenge in human health. Although it is known that genes causing similar diseases tend to lie close to one another in a network of protein-protein or functional interactions, the identification of these protein-protein networks is difficult to unravel. Here, we show that Msx1, Snail, Lhx6, Lhx8, Sp3, and Lef1 interact in vitro and in vivo, revealing the existence of a novel context-specific protein network. These proteins are all expressed in the neural crest-derived dental mesenchyme and cause tooth agenesis disorder when mutated in mouse and/or human. We also identified an in vivo direct target for Msx1 function, the cyclin D-dependent kinase (CDK) inhibitor p19(ink4d), whose transcription is differentially modulated by the protein network. Considering the important role of p19(ink4d) as a cell cycle regulator, these results provide evidence for the first time of the unique plasticity of the Msx1-dependent network of proteins in conferring differential transcriptional output and in controlling the cell cycle through the regulation of a cyclin D-dependent kinase inhibitor. Collectively, these data reveal a novel protein network operating in the neural crest-derived dental mesenchyme that is relevant for many other areas of developmental and evolutionary biology.

Msx1 and Tbx2 antagonistically regulate Bmp4 expression during the bud-to-cap stage transition in tooth development.

Bmp4 expression is tightly regulated during embryonic tooth development, with early expression in the dental epithelial placode leading to later expression in the dental mesenchyme. Msx1 is among several transcription factors that are induced by epithelial Bmp4 and that, in turn, are necessary for the induction and maintenance of dental mesenchymal Bmp4 expression. Thus, Msx1(-/-) teeth arrest at early bud stage and show loss of Bmp4 expression in the mesenchyme. Ectopic expression of Bmp4 rescues this bud stage arrest. We have identified Tbx2 expression in the dental mesenchyme at bud stage and show that this can be induced by epithelial Bmp4. We also show that endogenous Tbx2 and Msx1 can physically interact in mouse C3H10T1/2 cells. In order to ascertain a functional relationship between Msx1 and Tbx2 in tooth development, we crossed Tbx2 and Msx1 mutant mice. Our data show that the bud stage tooth arrest in Msx1(-/-) mice is partially rescued in Msx1(-/-);Tbx2(+/-) compound mutants. This rescue is accompanied by formation of the enamel knot (EK) and by restoration of mesenchymal Bmp4 expression. Finally, knockdown of Tbx2 in C3H10T1/2 cells results in an increase in Bmp4 expression. Together, these data identify a novel role for Tbx2 in tooth development and suggest that, following their induction by epithelial Bmp4, Msx1 and Tbx2 in turn antagonistically regulate odontogenic activity that leads to EK formation and to mesenchymal Bmp4 expression at the key bud-to-cap stage transition.

Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields.

Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical-free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5-100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10(5) -1.4 ± 0.2 × 10(5) cell/mL range. Our results suggest that IDPEFs may prove useful as a non-chemical method for fibroblast density control in human wound healing.

Developmental stalling and organ-autonomous regulation of morphogenesis.

Timing of organ development during embryogenesis is coordinated such that at birth, organ and fetal size and maturity are appropriately proportioned. The extent to which local developmental timers are integrated with each other and with the signaling interactions that regulate morphogenesis to achieve this end is not understood. Using the absolute requirement for a signaling pathway activity (bone morphogenetic protein, BMP) during a critical stage of tooth development, we show that suboptimal levels of BMP signaling do not lead to abnormal morphogenesis, as suggested by mutants affecting BMP signaling, but to a 24-h stalling of the intrinsic developmental clock of the tooth. During this time, BMP levels accumulate to reach critical levels whereupon tooth development restarts, accelerates to catch up with development of the rest of the embryo and completes normal morphogenesis. This suggests that individual organs can autonomously control their developmental timing to adjust their stage of development to that of other organs. We also find that although BMP signaling is critical for the bud-to-cap transition in all teeth, levels of BMP signaling are regulated differently in multicusped teeth. We identify an interaction between two homeodomain transcription factors, Barx1 and Msx1, which is responsible for setting critical levels of BMP activity in multicusped teeth and provides evidence that correlates the levels of Barx1 transcriptional activity with cuspal complexity. This study highlights the importance of absolute levels of signaling activity for development and illustrates remarkable self-regulation in organogenesis that ensures coordination of developmental processes such that timing is subordinate to developmental structure.

Molecular genetics of tooth development.

Organogenesis depends upon a well-ordered series of inductive events involving coordination of molecular pathways that regulate the generation and patterning of specific cell types. Key questions in organogenesis involve the identification of the molecular mechanisms by which proteins interact to organize distinct pattern formation and cell fate determination. Tooth development is an excellent context for investigating this complex problem because of the wealth of information emerging from studies of model organisms and human mutations. Since there are no obvious sources of stem cells in adult human teeth, any attempt to create teeth de novo will probably require the reprogramming of other cell types. Thus, the fundamental understanding of the control mechanisms responsible for normal tooth patterning in the embryo will help us understand cell fate specificity and may provide valuable information towards tooth organ regeneration.

Molecular genetics of ameloblast cell lineage.

Late tooth morphogenesis is characterized by a series of events that determine crown morphogenesis and the histodifferentiation of epithelial cells into enamel-secreting ameloblasts and of mesenchymal cells into dentin-secreting odontoblasts. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete a number of enamel-specific proteins. After depositing the full thickness of enamel matrix, ameloblasts shrink in size and regulate enamel maturation. Amelogenesis imperfecta (AI) is a heterogeneous group of inherited defects in enamel formation. Clinically, AI presents as a spectrum of enamel malformations that are categorized as hypoplastic, hypocalcified, or hypomaturation types, based upon the thickness and hardness of the enamel. The different types of AI are inherited, either as X-linked, autosomal-dominant, or autosomal-recessive traits. Recently, several gene mutations have been identified to cause the subtypes of AI. How these genes, however, coordinate their function to control amelogenesis is not understood. In this review, we discuss the role of genes that play definitive role on the determination of ameloblast cell fate and life cycle based on studies in transgenic animals.

Modification of Msx1 by SUMO-1.

The small ubiquitin-related modifier SUMO reversibly modifies many proteins, including promoter-specific transcription factors. Genetic studies in both humans and mice indicate that the Msx1 transcription factor is associated with specific disorders, including cleft palate. We show that Msx1 conjugation to SUMO-1 in vivo is enhanced by an E3 SUMO ligase, PIAS1, suggesting that sumoylation of Msx1 is a new mechanism for modulating the molecular function of Msx1 during organogenesis.

Msx2 controls ameloblast terminal differentiation.

Late tooth morphogenesis is characterized by a series of events that determine cusp morphogenesis and the histodifferentiation of epithelial cells into enamel-secreting ameloblasts. Mice lacking the homeobox gene Msx2 exhibit defects in cusp morphogenesis and in the process of amelogenesis. To better understand the basis of the Msx2 mutant tooth defects, we have investigated the function of Msx2 during late stages of tooth morphogenesis. Cusp formation is thought to be under the control of the enamel knot, which has been proposed to act as an organizing center during this process (Vaahtokari et al. [ 1996] Mech. Dev. 54:39-43). Bone morphogenetic protein-4 (BMP4) has been suggested to mediate termination of enamel knot signaling by means of regulation of programmed cell death (Jernvall et al. [ 1998] Development 125:161-169). Here, we show that Bmp4 expression in the enamel knot is Msx2-dependent. We further show that during amelogenesis Msx2 is required for the expression of the extracellular matrix gene Laminin 5 alpha 3, which is known to play an essential role during ameloblast differentiation. This result thus provides a paradigm for understanding how transcription factors and extracellular matrix can be integrated into a developmental pathway controlling cell differentiation.

Runx2 mediates FGF signaling from epithelium to mesenchyme during tooth morphogenesis.

Runx2 (Cbfa1) is a runt domain transcription factor that is essential for bone development and tooth morphogenesis. Teeth form as ectodermal appendages and their development is regulated by interactions between the epithelium and mesenchyme. We have shown previously that Runx2 is expressed in the dental mesenchyme and regulated by FGF signals from the epithelium, and that tooth development arrests at late bud stage in Runx2 knockout mice [Development 126 (1999) 2911]. In the present study, we have continued to clarify the role of Runx2 in tooth development and searched for downstream targets of Runx2 by extensive in situ hybridization analysis. The expression of Fgf3 was downregulated in the mesenchyme of Runx2 mutant teeth. FGF-soaked beads failed to induce Fgf3 expression in Runx2 mutant dental mesenchyme whereas in wild-type mesenchyme they induced Fgf3 in all explants indicating a requirement of Runx2 for transduction of FGF signals. Fgf3 was absent also in cultured Runx2-/- calvarial cells and it was induced by overexpression of Runx2. Furthermore, Runx2 was downregulated in Msx1 mutant tooth germs, indicating that it functions in the dental mesenchyme between Msx1 and Fgf3. Shh expression was absent from the epithelial enamel knot in lower molars of Runx2 mutant and reduced in upper molars. However, other enamel knot marker genes were expressed normally in mutant upper molars, while reduced or missing in lower molars. These differences between mutant upper and lower molars may be explained by the substitution of Runx2 function by Runx3, another member of the runt gene family that was upregulated in upper but not lower molars of Runx2 mutants. Shh expression in mutant enamel knots was not rescued by FGFs in vitro, indicating that in addition to Fgf3, Runx2 regulates other mesenchymal genes required for early tooth morphogenesis. Also, exogenous FGF and SHH did not rescue the morphogenesis of Runx2 mutant molars. We conclude that Runx2 mediates the functions of epithelial FGF signals regulating Fgf3 expression in the dental mesenchyme and that Fgf3 may be a direct target gene of Runx2.

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

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