Epithelial-mesenchymal Interactions Research Articles

Epithelial-Mesenchymal Interaction in Hair Regeneration and Skin Wound Healing.

Interactions between epithelial and mesenchymal cells influence hair follicles (HFs) during embryonic development and skin regeneration following injury. Exchanging soluble molecules, altering key pathways, and extracellular matrix signal transduction are all part of the interplay between epithelial and mesenchymal cells. In brief, the mesenchyme contains dermal papilla cells, while the hair matrix cells and outer root sheath represent the epithelial cells. This study summarizes typical epithelial–mesenchymal signaling molecules and extracellular components under the control of follicular stem cells, aiming to broaden our current understanding of epithelial–mesenchymal interaction mechanisms in HF regeneration and skin wound healing.

Tubular dentin formation by TGF-β/BMP signaling in dental epithelial cells.

This study aimed to identify formation of tubular dentin induced by transforming growth factor-β (TGF-β) and bone morphogenic protein (BMP) signaling pathway in dental epithelial cells. We collected conditioned medium (CM) of rTGF-β1/rBMP-2-treated HAT-7 and treated to MDPC-23 cells. The expression levels of odontoblast differentiation markers, KLF4, DMP1, and DSP were evaluated by real-time PCR and Western blot analysis. To evaluate whether CM of rTGF-β1/rBMP-2 induces tubular dentin formation, we made a beagle dog tooth defect model. Here, we show that Cpne7 is regulated by Smad4-dependent TGF-β1/BMP2 signaling pathway in dental epithelial cells. CM of rTGF-β1/rBMP-2 treated HAT-7 or rCPNE7 raises the expression levels of KLF4, DMP1, and DSP in MDPC-23 cells. When rTGF-β1 or rBMP-2 is directly treated to MDPC-23 cells, however, expression levels of Cpne7-regulated genes remain unchanged. In a beagle dog defect model, application of rTGF-β1/BMP2-treated CM resulted in tubular tertiary dentin mixed with osteodentin at cavity-prepared sites, while rTGF-β1 group exhibited homogenous osteodentin. Taken together, Smad4-dependent TGF-β1/BMP2 signaling regulates Cpne7 in dental epithelial cells, and CPNE7 protein secreted from pre-ameloblasts mediates odontoblast differentiation via epithelial-mesenchymal interaction.

Alginate Hydrogel Microtubes for Salivary Gland Cell Organization and Cavitation.

Understanding the different regulatory functions of epithelial and mesenchymal cell types in salivary gland development and cellular organization is essential for proper organoid formation and salivary gland tissue regeneration. Here, we demonstrate a biocompatible platform using pre-formed alginate hydrogel microtubes to facilitate direct epithelial–mesenchymal cell interaction for 3D salivary gland cell organization, which allows for monitoring cellular organization while providing a protective barrier from cell-cluster loss during medium changes. Using mouse salivary gland ductal epithelial SIMS cells as the epithelial model cell type and NIH 3T3 fibroblasts or primary E16 salivary mesenchyme cells as the stromal model cell types, self-organization from epithelial–mesenchymal interaction was examined. We observed that epithelial and mesenchymal cells undergo aggregation on day 1, cavitation by day 4, and generation of an EpCAM-expressing epithelial cell layer as early as day 7 of the co-culture in hydrogel microtubes, demonstrating the utility of hydrogel microtubes to facilitate heterotypic cell–cell interactions to form cavitated organoids. Thus, pre-formed alginate microtubes are a promising co-culture method for further understanding epithelial and mesenchymal interaction during tissue morphogenesis and for future practical applications in regenerative medicine.

Spatiotemporal Expression Patterns of Critical Genes Involved in FGF Signaling During Morphogenesis and Odontogenesis of Deciduous Molars in Miniature Pigs.

The fibroblast growth factor (FGF) pathway plays an important role in epithelial-mesenchymal interactions during tooth development. Nevertheless, how the ligands, receptors, and antagonists of the FGF pathway are involved in epithelial-mesenchymal interactions remains largely unknown. Miniature pigs exhibit tooth anatomy and replacement patterns like those in humans and hence can serve as large animal models. The present study investigated the spatiotemporal expression patterns of critical genes encoding FGF ligands (FGF3, FGF4, FGF7, and FGF9), antagonists (SPRY2 and SPRY4) and receptors (FGFR1, FGFR2, and FGFR3) in the third deciduous molars of miniature pigs at the cap (embryonic day 40, E40), early bell (E50), and late bell (E60) stages. The results of in situ hybridization (ISH) with tyramide signal amplification and of qRT-PCR analysis revealed increased expression of FGF7, FGFR1, FGFR2, and SPRY4 in dental epithelium and of FGF7 and FGFR1 in mesenchyme from E40 to E50. In contrast, the results revealed decreased expression of FGF3, FGF4, FGF9, and FGFR3 in dental epithelium and of FGF4, FGF9, FGFR2, and FGFR3 in the mesenchyme from E40 to E60. Mesenchyme signals of FGF3, FGF4, FGF7, SPRY2, FGFR2, and FGFR3 were concentrated in the odontoblast layer from E50 to E60. The distinct expression patterns of these molecules indicated elaborate regulation during dental morphogenesis. Our results provide a foundation for further investigation into fine-tuning dental morphogenesis and odontogenesis by controlling interactions between dental epithelium and mesenchyme, thus promoting tooth regeneration in large mammals.

Spatiotemporal Characteristics of Rectal Carcinoma Cell Epithelial Mesenchymal Transition and Interaction with Immune Cells

Spatiotemporal Characteristics of Rectal Carcinoma Cell Epithelial Mesenchymal Transition and Interaction with Immune Cells

Epithelial Wntless regulates postnatal alveologenesis.

Alveologenesis requires the coordinated modulation of the epithelial and mesenchymal compartments to generate mature alveolar saccules for efficient gas exchange. However, the molecular mechanisms underlying the epithelial-mesenchymal interaction during alveologenesis are poorly understood. Here, we report that Wnts produced by epithelial cells are crucial for neonatal alveologenesis. Deletion of the Wnt chaperone protein Wntless homolog (Wls) disrupts alveolar formation, resulting in enlarged saccules in Sftpc-Cre/Nkx2.1-Cre; Wlsloxp/loxp mutants. Although commitment of the alveolar epithelium is unaffected, α-SMA+ mesenchymal cells persist in the alveoli, accompanied by increased collagen deposition, and mutants exhibit exacerbated fibrosis following bleomycin challenge. Notably, α-SMA+ cells include a significant number of endothelial cells resembling endothelial to mesenchymal transition (EndMT), which is also present in Ager-CreER; Wlsloxp/loxp mutants following early postnatal Wls deletion. These findings provide initial evidence that epithelial-derived Wnts are crucial for the differentiation of the surrounding mesenchyme during early postnatal alveologenesis.

Roles of cutaneous cell-cell communication in wound healing outcome: An emphasis on keratinocyte-fibroblast crosstalk.

Tissue repair is a very complex event and involves a continuously orchestrated sequence of signals and responses from platelets, fibroblasts, epithelial, endothelial and immune cells. The details of interaction between these signals, which are mainly growth factors and cytokines, have been widely discussed. However, it is still not clear how activated cells at wound sites lessen their activities after epithelialization is completed. Termination of the wound healing process requires a fine balance between extracellular matrix (ECM) deposition and degradation. Maintaining this balance requires highly accurate epithelial-mesenchymal communication and correct information exchange between keratinocytes and fibroblasts. As it has been reported in the literature, a disruption in epithelialization during the process of wound healing increases the frequency of developing chronic wounds or fibrotic conditions, as seen in a variety of clinical cases. Conversely, the potential stop signal for wound healing should have a regulatory role on both ECM synthesis and degradation to reach a successful wound healing outcome. This review briefly describes the potential roles of growth factors and cytokines in controlling the early phase of wound healing and predominantly explores the role of releasable factors from epithelial-mesenchymal interaction in controlling during and the late stage of the healing process. Emphasis will be given on the crosstalk between keratinocytes and fibroblasts in ECM modulation and the healing outcome following a brief discussion of the wound healing initiation mechanism. In particular, we will review the termination of acute dermal wound healing, which frequently leads to the development of hypertrophic scarring.

Engineering Epithelial-Mesenchymal Microtissues to Study Cell-Cell Interactions in Development.

Intercellular signaling drives human development, but there is a paucity of in vitro models that recapitulate important tissue architecture while remaining operationally simple and scalable. As an example, formation of the upper lip and palate requires the orchestrated proliferation and fusion of embryonic facial growth centers and is dependent on paracrine epithelial-mesenchymal signaling through multiple pathways including the Sonic Hedgehog (SHH), transforming growth factor-beta (Tgf-β), bone morphogenic protein (BMP), and epidermal growth factor (EGF) pathways. We have developed a robust, throughput-compatible microphysiological system to model intercellular signaling including epithelial-mesenchymal interactions that is useful for studying both normal and abnormal orofacial development. We describe the construction and operation of an engineered microplate created using CNC micromilling of 96-well microtiter plates capable of containing up to 20 epithelial-mesenchymal microtissues. A dense three-dimensional mesenchyme is created by embedding cells (O9-1, 3T3) in a biomimetic hydrogel. An epithelial layer is then overlayed on the microtissue by loading cells in engineered microchannels that flank the microtissue. The result is an engineering epithelial-mesenchymal interface that is both on and perpendicular to the imaging plane making it suitable for high-content imaging and analysis. The resulting microtissues and device are compatible with diverse analytical techniques including fluorescent and luminescent cell health and enzymatic reporter assays, gene expression analyses, and protein staining. This tractable model and approach promise to shed light on critical processes in intercellular signaling events in orofacial development and beyond.

Disease modeling of pulmonary fibrosis using human pluripotent stem cell-derived alveolar organoids.

SummaryAlthough alveolar epithelial cells play a critical role in the pathogenesis of pulmonary fibrosis, few practical in vitro models exist to study them. Here, we established a novel in vitro pulmonary fibrosis model using alveolar organoids consisting of human pluripotent stem cell-derived alveolar epithelial cells and primary human lung fibroblasts. In this human model, bleomycin treatment induced phenotypes such as epithelial cell-mediated fibroblast activation, cellular senescence, and presence of alveolar epithelial cells in abnormal differentiation states. Chemical screening performed to target these abnormalities showed that inhibition of ALK5 or blocking of integrin αVβ6 ameliorated the fibrogenic changes in the alveolar organoids. Furthermore, organoid contraction and extracellular matrix accumulation in the model recapitulated the pathological changes observed in pulmonary fibrosis. This human model may therefore accelerate the development of highly effective therapeutic agents for otherwise incurable pulmonary fibrosis by targeting alveolar epithelial cells and epithelial-mesenchymal interactions.

Isolation of Epithelial Cells from Human Dental Follicle.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while α-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells. The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

Spatiotemporal Changes in Transcriptome of Odontogenic and Non-odontogenic Regions in the Dental Arch of Mus musculus.

Over the past 40 years, studies on tooth regeneration have been conducted. These studies comprised two main flows: some focused on epithelial–mesenchymal interaction in the odontogenic region, whereas others focused on creating a supernumerary tooth in the non-odontogenic region. Recently, the scope of the research has moved from conventional gene modification and molecular therapy to genome and transcriptome sequencing analyses. However, these sequencing data have been produced only in the odontogenic region. We provide RNA-Seq data of not only the odontogenic region but also the non-odontogenic region, which loses tooth-forming capacity during development and remains a rudiment. Sequencing data were collected from mouse embryos at three different stages of tooth development. These data will expand our understanding of tooth development and will help in designing developmental and regenerative studies from a new perspective.

Restoration of Immune Privilege in Human Dermal Papillae Controlling Epithelial-Mesenchymal Interactions in Hair Formation.

Hair follicles are among a handful of organs that exhibit immune privilege. Dysfunction of the hair follicle immune system underlies the development of inflammatory diseases, such as alopecia areata. Quantitative reverse transcription PCR and immunostaining was used to confirm the expression of major histocompatibility complex class I in human dermal papilla cells. Through transcriptomic analyses of human keratinocyte stem cells, major histocompatibility complex class I was identified as differentially expressed genes. Organ culture and patch assay were performed to assess the ability of WNT3a conditioned media to rescue immune privilege. Lastly, CD8+ T cells were detected near the hair bulb in alopecia areata patients through immunohistochemistry. Inflammatory factors such as tumor necrosis factor alpha and interferon gamma were verified to induce the expression of major histocompatibility complex class I proteins in dermal papilla cells. Additionally, loss of immune privilege of hair follicles was rescued following treatment with conditioned media from outer root sheath cells. Transcriptomic analyses found 58 up-regulated genes and 183 down-regulated genes related in MHC class I+ cells. Using newborn hairpatch assay, we demonstrated that WNT3a conditioned media with epidermal growth factor can restore hair growth. In alopecia areata patients, CD8+ T cells were increased during the transition from mid-anagen to late catagen. Identification of mechanisms governing epithelial and mesenchymal interactions of the hair follicle facilitates an improved understanding of the regulation of hair follicle immune privilege.

Microstructured Hyaluronic Acid Hydrogel for Tooth Germ Bioengineering.

Tooth loss has been found to adversely affect not just masticatory and speech functions, but also psychological health and quality of life. Currently, teeth replacement options include dentures, bridges, and implants. However, these artificial replacement options remain inferior to biological replacements due to their reduced efficiency, the need for replacements, and the risk of immunological rejection. To this end, there has been a heightened interest in the bioengineering of teeth in recent years. While there have been reports of successfully regenerated teeth, controlling the size and shape of bioengineered teeth remains a challenge. In this study, methacrylated hyaluronic acid (MeHA) was synthesized and microstructured in a hydrogel microwell array using soft lithography. The resulting MeHA hydrogel microwell scaffold resembles the shape of a naturally developing human tooth germ. To facilitate the epithelial–mesenchymal interactions, human adult low calcium high temperature (HaCaT) cells were seeded on the surface of the hydrogels and dental pulp stem cells (DPSCs) were encapsulated inside the hydrogels. It was found that hydrogel scaffolds were able to preserve the viability of both types of cells and they appeared to favor signaling between epithelial and mesenchymal cells, which is necessary in the promotion of cell proliferation. As such, the hydrogel scaffolds offer a promising system for the bioengineering of human tooth germs in vitro.

Cell Differentiation and Replication during Postnatal Development of the Murine First Molar.

Simple SummaryTeeth are necessary to prepare food for swallowing. The process of teeth development before and after birth may be studied in normal mice and also by reproducing diseases or genetic conditions. However, mice teeth are different from human teeth, since mice have only permanent teeth. Moreover, their incisors continue to grow for the whole lifespan. Hence, it is important to know how the mouse teeth develop. We studied the development of the first molar in mice from birth to weaning and showed that dividing cells are located in a different part of the developing tooth according to age.Various signaling molecular pathways are involved in odontogenesis to promote cellular replication and differentiation. Tooth formation is controlled mainly by epithelial–mesenchymal interactions. The aim of this work was to investigate how cellular replication and differentiation ensue during the formation of the murine first molar in postnatal ages until eruption, focusing on morphogenesis, odontoblast differentiation and cellular replication. Wild-type CD1 mice were examined from birth to weaning. Morphogenesis and interaction between developing epithelial and mesenchymal tissues were evaluated in hematoxylin–eosin and Gomori trichome stained sections. Immunohistochemistry for nestin, which mediates the differentiation of odontoblasts, especially their polarization and elongation, showed that this intermediate filament was apparent already at postnatal day P1 in the apical region of odontoblasts and progressed apically from cusp tips, while it was not present in epithelial tissues. The expression of nuclear antigen Ki-67 highlighted dividing cells in both epithelial and mesenchymal tissues at P1, while one week later they were restricted to the cementoenamel junction, guiding root elongation. The link between odontoblast maturation and cellular replication in the different tooth tissues is essential to understand the development of tooth shape and dimension, to outline mechanisms of tooth morphogenesis and possibly eruption.

Development of a Human Intestinal Organoid Model for In Vitro Studies on Gut Inflammation and Fibrosis.

Inflammatory Bowel Diseases (IBDs) are characterized by chronic intestinal inflammation and fibrosis, the latter being the predominant denominator for long-term complications. Epithelial and mesenchymal 2D cultures are highly utilized in vitro models for the preclinical evaluation of anti-inflammatory and antifibrotic therapies. More recently, human intestinal organoids (HIOs), a new 3D in vitro model derived from pluripotent stem cells, have the advantage to closely resemble the architecture of the intestinal mucosa. However, the appropriate timing for the study of inflammatory and fibrotic responses, during HIO development, has not been adequately investigated. We developed HIOs from the human embryonic stem cell line, H1, and examined the expression of mesenchymal markers during their maturation process. We also investigated the effect of inflammatory stimuli on the expression of fibrotic and immunological mediators. Serial evaluation of the expression of mesenchymal and extracellular matrix (ECM) markers revealed that HIOs have an adequately developed mesenchymal component, which gradually declines through culture passages. Specifically, CD90, collagen type I, collagen type III, and fibronectin were highly expressed in early passages but gradually diminished in late passages. The proinflammatory cytokines IL-1α and TNF-α induced the mRNA expression of fibronectin, collagen types I and III, tissue factor (TF), and alpha-smooth muscle actin (α-SMA) primarily in early passages. Similarly, HIOs elicited strong mRNA and protein mesenchymal (CXCL10) and epithelial (CXCL1, CCL2, CXCL8, and CCL20) chemokine responses in early but not late passages. In contrast, the epithelial tight junction components, CLDN1 and JAMA, responded to inflammatory stimulation independently of the culture passage. Our findings indicate that this HIO model contains a functional mesenchymal component, during early passages, and underline the significance of the mesenchymal cells' fitness in inflammatory and fibrotic responses. Therefore, we propose that this model is suitable for the study of epithelial-mesenchymal interactions in early passages when the mesenchymal component is active.

Lung branching morphogenesis is accompanied by temporal metabolic changes towards a glycolytic preference

BackgroundLung branching morphogenesis is characterized by epithelial-mesenchymal interactions that ultimately define the airway conducting system. Throughout this process, energy and structural macromolecules are necessary to sustain the high proliferative rates. The extensive knowledge of the molecular mechanisms underlying pulmonary development contrasts with the lack of data regarding the embryonic lung metabolic requirements. Here, we studied the metabolic profile associated with the early stages of chicken pulmonary branching.MethodsIn this study, we used an ex vivo lung explant culture system and analyzed the consumption/production of extracellular metabolic intermediates associated with glucose catabolism (alanine, lactate, and acetate) by 1H-NMR spectroscopy in the culture medium. Then, we characterized the transcript levels of metabolite membrane transporters (glut1, glut3, glut8, mct1, mct3, mct4, and mct8) and glycolytic enzymes (hk1, hk2, pfk1, ldha, ldhb, pdha, and pdhb) by qPCR. ldha and ldhb mRNA spatial localization was determined by in situ hybridization. Proliferation was analyzed by directly assessing DNA synthesis using an EdU-based assay. Additionally, we performed western blot to analyze LDHA and LDHT protein levels. Finally, we used a Clark-Type Electrode to assess the lung explant's respiratory capacity.ResultsGlucose consumption decreases, whereas alanine, lactate, and acetate production progressively increase as branching morphogenesis proceeds. mRNA analysis revealed variations in the expression levels of key enzymes and transporters from the glycolytic pathway. ldha and ldhb displayed a compartment-specific expression pattern that resembles proximal–distal markers. In addition, high proliferation levels were detected at active branching sites. LDH protein expression levels suggest that LDHB may account for the progressive rise in lactate. Concurrently, there is a stable oxygen consumption rate throughout branching morphogenesis.ConclusionsThis report describes the temporal metabolic changes that accompany the early stages of chicken lung branching morphogenesis. Overall, the embryonic chicken lung seems to shift to a glycolytic lactate-based metabolism as pulmonary branching occurs. Moreover, this metabolic rewiring might play a crucial role during lung development.

DSPP dosage affects tooth development and dentin mineralization.

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

Charting human development using a multi-endodermal organ atlas and organoid models

SummaryOrgans are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Expression and localization of FGFR1, FGFR2 and CTGF during normal human lung development

Aim of our study was to provide insight into the temporal and spatial expression of FGFR1, FGFR2 and CTGF during normal human lung development which may have an important impact on understanding occurrence of developmental lung anomalies. Morphological parameters were analysed using double immunofluorescence on human embryonal (6th and 7th developmental week-dw) and foetal (8th, 9th and 16th developmental week) human lung samples.FGFR1 and FGFR2 was positive during all the dw in both the epithelium and mesenchyme. The highest number of FGFR1 positive cells was observed during the 6th dw (112/mm2) and 9th dw (87/mm2) in the epithelium compared to the 7th, 8th and 16th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001). The highest number of FGFR1 positive cells in the mesenchyme was observed during the 8th dw (19/mm2) and 16th dw (13/mm2) compared to the 6th, 7th, and 9th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001). The number of FGFR1 positive cells in the epithelium was higher for FGFR2 compared to number of positive cells (Mann-Whitney test, p < 0.0001). FGFR2 showed the highest number in the epithelium during the 7th dw (111/mm2) and 9th dw (87/mm2) compared to 6th, 8th and 16th dw (Kruskal-Wallis test, p < 0.001, p < 0.0001, p < 0.01 respectively). The highest number of FGFR2 positive cells in the mesenchyme was observed during the 9th dw (26/mm2), compared to the 6th, 7th,8th and 16th dw (Kruskal-Wallis test, p < 0.0001), while the number of FGFR2 positive cells in the epithelium was significantly higher than in the mesenchyme (Mann-Whitney test, p < 0.0001). CTGF was negative in both epithelium and mesenchyme during all except the 16th dw in the mesenchyme where it co-localized with FGFR2.FGFR1 and FGFR2 might be essential for epithelial-mesenchymal interactions that determine epithelial branching and mesenchymal growth during early lung development. Sudden increase in FGF1 in the epithelium and FGF2 in the mesenchyme in the foetus at 9th dw could be associated with the onset of foetal breathing movements. CTGF first appear during the foetal lung development.

Transcriptional regulation of the basichelix-loop-helix factor AmeloD during tooth development.

The epithelial-mesenchymal interactions are essential for the initiation and regulation of the development of teeth. Following the initiation of tooth development, numerous growth factors are secreted by the dental epithelium and mesenchyme that play critical roles in cellular differentiation. During tooth morphogenesis, the dental epithelial stem cells differentiate into several cell types, including inner enamel epithelial cells, which then differentiate into enamel matrix-secreting ameloblasts. Recently, we reported that the novel basic-helix-loop-helix transcription factor, AmeloD, is actively engaged in the development of teeth as a regulator of dental epithelial cell motility. However, the gene regulation mechanism of AmeloD is still unknown. In this study, we aimed to uncover the mechanisms regulating AmeloD expression during tooth development. By screening growth factors that are important in the early stages of tooth formation, we found that TGF-β1 induced AmeloD expression and ameloblast differentiation in the dental epithelial cell line, SF2. TGF-β1 phosphorylated ERK1/2 and Smad2/3 to induce AmeloD expression, whereas treatment with the MEK inhibitor, U0126, inhibited AmeloD induction. Promoter analysis of AmeloD revealed that the proximal promoter of AmeloD showed high activity in dental epithelial cell lines, which was enhanced following TGF-β1 stimulation. These results suggested that TGF-β1 activates AmeloD transcription via ERK1/2 phosphorylation. Our findings provide new insights into the mechanisms that govern tooth development.