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1.
Dis Model Mech ; 2024 Jun 21.
Article in English | MEDLINE | ID: mdl-38903015

ABSTRACT

Structural changes to vocal fold (VF) epithelium, namely loosened intercellular junctions have been reported in VF benign lesions. Potential mechanisms responsible for the disruption of cell junctions do not address the contribution of resident microbial communities to this pathological phenomenon. In this study, we focused on determining the relationship between Streptococcus pseudopneumoniae (SP), a dominant bacterial species associated with benign lesions, and S. salivarius (SS), a commensal bacterium, with human VF epithelial cells, in our three-dimensional model of human VF mucosa. This experimental system enabled direct deposition of bacteria onto constructs at the Air/Liquid interface allowing for the assessment of bacteria-host interactions at cellular, molecular and ultrastructural levels. Our findings demonstrate that SP disrupts VF epithelial integrity and initiates inflammation via exported products, HtrA1 and pneumolysin. In contrast, SS attaches to VF epithelium, reduces inflammation and induces Mmp2-mediated apical desquamation of infected cells to mitigate the impact of pathogens. In conclusion, this study highlights the complexity of microbial involvement in VF pathology and potential VF mucosal restoration in the presence of laryngeal commensals.

2.
Cell Mol Life Sci ; 79(12): 591, 2022 Nov 14.
Article in English | MEDLINE | ID: mdl-36376494

ABSTRACT

Mechanoreceptors are implicated as functional afferents within mucosa of the airways and the recent discovery of mechanosensitive channels Piezo1 and Piezo2 has proved essential for cells of various mechanically sensitive tissues. However, the role for Piezo1/2 in vocal fold (VF) mucosal epithelia, a cell that withstands excessive biomechanical insult, remains unknown. The purpose of this study was to test the hypothesis that Piezo1 is required for VF mucosal repair pathways of epithelial cell injury. Utilizing a sonic hedgehog (shh) Cre line for epithelial-specific ablation of Piezo1/2 mechanoreceptors, we investigated 6wk adult VF mucosa following naphthalene exposure for repair strategies at 1, 3, 7 and 14 days post-injury (dpi). PIEZO1 localized to differentiated apical epithelia and was paramount for epithelial remodeling events. Injury to wildtype epithelium was most appreciated at 3 dpi. Shhcre/+; Piezo1loxP/loxP, Piezo2 loxP/+ mutant epithelium exhibited severe cell/nuclear defects compared to injured controls. Conditional ablation of Piezo1 and/or Piezo2 to uninjured VF epithelium did not result in abnormal phenotypes across P0, P15 and 6wk postnatal stages compared to heterozygote and control tissue. Results demonstrate a role for Piezo1-expressing VF epithelia in regulating self-renewal via effects on p63 transcription and YAP subcellular translocation-altering cytokeratin differentiation.


Subject(s)
Hedgehog Proteins , Keratins , Hedgehog Proteins/metabolism , Keratins/genetics , Vocal Cords/metabolism , Epithelial Cells/metabolism , Epithelium/metabolism
3.
Front Cell Dev Biol ; 10: 942622, 2022.
Article in English | MEDLINE | ID: mdl-35938172

ABSTRACT

The larynx, trachea, and esophagus share origin and proximity during embryonic development. Clinical and experimental evidence support the existence of neurophysiological, structural, and functional interdependencies before birth. This investigation provides the first comprehensive transcriptional profile of all three organs during embryonic organogenesis, where differential gene expression gradually assembles the identity and complexity of these proximal organs from a shared origin in the anterior foregut. By applying bulk RNA sequencing and gene network analysis of differentially expressed genes (DEGs) within and across developing embryonic mouse larynx, esophagus, and trachea, we identified co-expressed modules of genes enriched for key biological processes. Organ-specific temporal patterns of gene activity corresponding to gene modules within and across shared tissues during embryonic development (E10.5-E18.5) are described, and the laryngeal transcriptome during vocal fold development and maturation from birth to adulthood is characterized in the context of laryngeal organogenesis. The findings of this study provide new insights into interrelated gene sets governing the organogenesis of this tripartite organ system within the aerodigestive tract. They are relevant to multiple families of disorders defined by cardiocraniofacial syndromes.

4.
Dis Model Mech ; 15(8)2022 08 01.
Article in English | MEDLINE | ID: mdl-35770504

ABSTRACT

Vaping has been reported to cause acute epiglottitis, a life-threatening airway obstruction induced by direct epithelial injury and subsequent inflammatory reaction. Here, we show that we were able to recapitulate this phenomenon in vitro. Exposure of human engineered vocal fold (VF) mucosae to 0.5% and 5% electronic cigarette (e-cigarette) vapor extract (ECVE) for 1 week induced cellular damage of luminal cells, disrupting homeostasis and innate immune responses. Epithelial erosion was likely caused by accumulation of solvents and lipid particles in the cytosol and intercellular spaces, which altered lipid metabolism and plasma membrane properties. Next, we investigated how the mucosal cells responded to the epithelial damage. We withdrew the ECVE from the experimental system and allowed VF mucosae to regenerate for 1, 3 and 7 days, which triggered intense epithelial remodeling. The epithelial changes included expansion of P63 (TP63)-positive basal cells and cytokeratin 14 (KRT14) and laminin subunit α-5 (LAMA5) deposition, which might lead to local basal cell hyperplasia, hyperkeratinization and basement membrane thickening. In summary, vaping presents a threat to VF mucosal health and airway protection, thereby raising further concerns over the safety of e-cigarette use. This article has an associated First Person interview with the first author of the paper.


Subject(s)
E-Cigarette Vapor , Electronic Nicotine Delivery Systems , Vaping , Humans , Mucous Membrane , Vaping/adverse effects , Vocal Cords
5.
Methods Mol Biol ; 2454: 365-380, 2022.
Article in English | MEDLINE | ID: mdl-33959913

ABSTRACT

Healthy vocal fold mucosa is composed of two major cell types, non-keratinized stratified squamous epithelium and vocal fold fibroblasts. Although dysfunction of the epithelium may play a significant pathogenic role in vocal fold diseases, studies at the genetic and molecular level using primary epithelial cells or models of human vocal fold mucosa have been significantly limited by the availability of relevant tissue types, poor growth, and heterogeneity of primary vocal fold epithelial cells. Here, we describe in vitro developmental differentiation of human induced pluripotent stem cells into vocal fold basal epithelial progenitors that were reseeded on collagen-fibroblast constructs to induce stratification and generate a three-dimensional model of human vocal fold mucosa. The engineered vocal fold mucosa represents physiologically relevant and clinically useful model that can be used as a tool for disease modeling and testing of therapeutic approaches for the treatment of laryngeal and VF inflammation.


Subject(s)
Induced Pluripotent Stem Cells , Cell Differentiation , Epithelial Cells , Humans , Laryngeal Mucosa , Vocal Cords/physiology
6.
PLoS One ; 16(1): e0245073, 2021.
Article in English | MEDLINE | ID: mdl-33439907

ABSTRACT

Current research approaches employ traditional tissue engineering strategies to promote vocal fold (VF) tissue regeneration, whereas recent novel advances seek to use principles of developmental biology to guide tissue generation by mimicking native developmental cues, causing tissue or allogenic/autologous progenitor cells to undergo the regeneration process. To address the paucity of data to direct VF differentiation and subsequent new tissue formation, we characterize structure-proliferation relationships and tissue elastic moduli over embryonic development using a murine model. Growth, cell proliferation, and tissue biomechanics were taken at E13.5, E15.5, E16.5, E18.5, P0, and adult time points. Quadratic growth patterns were found in larynx length, maximum transverse diameter, outer dorsoventral diameter, and VF thickness; internal VF length was found to mature linearly. Cell proliferation measured with EdU in the coronal and transverse planes of the VFs was found to decrease with increasing age. Exploiting atomic force microscopy, we measured significant differences in tissue stiffness across all time points except between E13.5 and E15.5. Taken together, our results indicate that as the VF mature and develop quadratically, there is a concomitant tissue stiffness increase. Greater gains in biomechanical stiffness at later prenatal stages, correlated with reduced cell proliferation, suggest that extracellular matrix deposition may be responsible for VF thickening and increased biomechanical function, and that the onset of biomechanical loading (breathing) may also contribute to increased stiffness. These data provide a profile of VF biomechanical and growth properties that can guide the development of biomechanically-relevant scaffolds and progenitor cell differentiation for VF tissue regeneration.


Subject(s)
Cell Differentiation , Cell Proliferation , Embryo, Mammalian/embryology , Embryonic Development , Extracellular Matrix/metabolism , Vocal Cords/embryology , Animals , Embryo, Mammalian/cytology , Mice , Vocal Cords/cytology
7.
FASEB J ; 35(2): e21243, 2021 02.
Article in English | MEDLINE | ID: mdl-33428261

ABSTRACT

Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVFE) lines for use in appropriate model systems. We steadily transfected a retroviral construct containing human telomerase reverse transcriptase (hTERT) into primary normal hVFE to establish a continuously replicating hVFE cell line. Immortalized hVFE across passages have cobblestone morphology, express epithelial markers cytokeratin 4, 13 and 14, induced hTERT gene and protein expression, have similar RNAseq profiling, and can continuously grow for more than 8 months. DNA fingerprinting and karyotype analysis demonstrated that immortalized hVFE were consistent with the presence of a single cell line. Validation of the hVFE, in a three-dimensional in vitro VF mucosal construct revealed a multilayered epithelial structure with VF epithelial cell markers. Wound scratch assay revealed higher migration capability of the immortalized hVFE on the surface of collagen-fibronectin and collagen gel containing human vocal fold fibroblasts (hVFF). Collectively, our report demonstrates the first immortalized hVFE from true VFs providing a novel and invaluable tool for the study of epithelial cell-fibroblast interactions that dictate disease and health of this specialized tissue.


Subject(s)
Epithelial Cells/cytology , Laryngeal Mucosa/cytology , Primary Cell Culture/methods , Vocal Cords/cytology , Aged , Cell Line , Cell Line Authentication/methods , Cell Proliferation , Cells, Cultured , Epithelial Cells/metabolism , Epithelial Cells/physiology , Female , Humans , Keratins/genetics , Keratins/metabolism , Male , Telomerase/genetics , Telomerase/metabolism
8.
Dev Biol ; 473: 33-49, 2021 05.
Article in English | MEDLINE | ID: mdl-33515576

ABSTRACT

Proliferation and differentiation of vocal fold epithelial cells during embryonic development is poorly understood. We examined the role of Hippo signaling, a vital pathway known for regulating organ size, in murine laryngeal development. Conditional inactivation of the Hippo kinase genes Lats1 and Lats2, specifically in vocal fold epithelial cells, resulted in severe morphogenetic defects. Deletion of Lats1 and Lats2 caused abnormalities in epithelial differentiation, epithelial lamina separation, cellular adhesion, basement membrane organization with secondary failed cartilage, and laryngeal muscle development. Further, Lats1 and Lats2 inactivation led to failure in differentiation of p63+ basal progenitors. Our results reveal novel roles of Hippo-Lats-YAP signaling in proper regulation of VF epithelial fate and larynx morphogenesis.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Larynx/physiology , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/physiology , Animals , Cell Cycle Proteins/metabolism , Cell Differentiation , Cell Proliferation/physiology , Epithelial Cells/metabolism , Epithelium/physiology , Female , Hippo Signaling Pathway , Larynx/metabolism , Male , Mice , Mice, Inbred C57BL , Morphogenesis , Protein Serine-Threonine Kinases/physiology , Signal Transduction/physiology , Transcription Factors/metabolism , Tumor Suppressor Proteins/physiology , Vocal Cords/metabolism , Vocal Cords/physiology , YAP-Signaling Proteins
9.
Dev Biol ; 466(1-2): 47-58, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32777221

ABSTRACT

In the present study, we investigated the role of mechanical load as generated by amniotic fluid in the vocal fold embryogenesis. In utero, amniotic fluid flows through the laryngeal inlet down into the lungs during fetal breathing and swallowing. In a mouse model, the onset of fetal breathing coincides with epithelial lamina recanalization. The epithelial lamina is a temporal structure that is formed during early stages of the larynx development and is gradually resorbed whereby joining the upper and lower airways. Here, we show that a temporary decrease in mechanical load secondary to drainage of amniotic fluid and subsequent flow restoration, impaired timing of epithelial lamina disintegration. Moreover, re-accumulation of fluid in the laryngeal region led to VF tissue deformation triggering remodeling of the epithelium and pressure generated changes in the elastic properties of the lamina propria, as measured by atomic force microscopy. We further show that load-related structural changes were likely mediated by Piezo 1 -Yap signaling pathway in the vocal fold epithelium. Understanding the relationship between the mechanical and biological parameters in the larynx is key to gaining insights into pathogenesis of congenital laryngeal disorders as well as mechanisms of vocal fold tissue remodeling in response to mechanotransduction.


Subject(s)
Amniotic Fluid/metabolism , Laryngeal Mucosa/embryology , Signal Transduction , Vocal Cords/embryology , Animals , Mice
10.
Cell Mol Life Sci ; 77(19): 3781-3795, 2020 Oct.
Article in English | MEDLINE | ID: mdl-32253462

ABSTRACT

The larynx and vocal folds sit at the crossroad between digestive and respiratory tracts and fulfill multiple functions related to breathing, protection and phonation. They develop at the head and trunk interface through a sequence of morphogenetic events that require precise temporo-spatial coordination. We are beginning to understand some of the molecular and cellular mechanisms that underlie critical processes such as specification of the laryngeal field, epithelial lamina formation and recanalization as well as the development and differentiation of mesenchymal cell populations. Nevertheless, many gaps remain in our knowledge, the filling of which is essential for understanding congenital laryngeal disorders and the evaluation and treatment approaches in human patients. This review highlights recent advances in our understanding of the laryngeal embryogenesis. Proposed genes and signaling pathways that are critical for the laryngeal development have a potential to be harnessed in the field of regenerative medicine.


Subject(s)
Laryngeal Diseases/pathology , Larynx/metabolism , Vocal Cords/metabolism , Animals , Cell Differentiation , Humans , Laryngeal Diseases/metabolism , Larynx/growth & development , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , SOXB1 Transcription Factors/metabolism , Signal Transduction , Thyroid Nuclear Factor 1/metabolism , Vocal Cords/growth & development
11.
Nat Commun ; 10(1): 4161, 2019 09 24.
Article in English | MEDLINE | ID: mdl-31551422

ABSTRACT

Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells' limited proliferative capacity and absence of cell lines. Here we report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa. Furthermore, we demonstrate mucosal inflammation upon exposure of these constructs to 5% cigarette smoke extract. Upregulation of pro-inflammatory genes in epithelium and fibroblasts leads to aberrant VF mucosa remodeling. Collectively, our results demonstrate that hiPSC-derived VF mucosa is a versatile tool for future investigation of genetic and molecular mechanisms underlying epithelium-fibroblasts interactions in health and disease.


Subject(s)
Induced Pluripotent Stem Cells/cytology , Mucous Membrane/growth & development , Smoking/adverse effects , Vocal Cords/growth & development , Cell Differentiation , Cell Line , Cells, Cultured , Endoderm/physiology , Fibroblast Growth Factors/metabolism , Gene Expression Regulation , Genes, Reporter , Genome , Green Fluorescent Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation/genetics , Inflammation/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Tissue Engineering
12.
Development ; 145(4)2018 02 16.
Article in English | MEDLINE | ID: mdl-29386246

ABSTRACT

Congenital laryngeal webs result from failure of vocal fold separation during development in utero Infants present with life-threatening respiratory problems at birth, and extensive lifelong difficulties in breathing and voicing. The molecular mechanisms that instruct vocal fold formation are rarely studied. Here, we show, for the first time, that conditional inactivation of the gene encoding ß-catenin in the primitive laryngopharyngeal epithelium leads to failure in separation of the vocal folds, which approximates the gross phenotype of laryngeal webbing. These defects can be traced to a series of morphogenesis defects, including delayed fusion of the epithelial lamina and formation of the laryngeal cecum, failed separation of the larynx and esophagus with reduced and disorganized cartilages and muscles. Parallel to these morphogenesis defects, inactivation of ß-catenin disrupts stratification of epithelial cells and establishment of p63+ basal progenitors. These findings provide the first line of evidence that links ß-catenin function to the cell proliferation and progenitor establishment during larynx and vocal fold development.


Subject(s)
Congenital Abnormalities/genetics , Larynx/abnormalities , Larynx/metabolism , Stem Cells/metabolism , Vocal Cords/metabolism , beta Catenin/metabolism , Animals , Cell Differentiation , Cell Proliferation , Fluorescent Antibody Technique , Mice
13.
Dev Biol ; 409(2): 429-41, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26632490

ABSTRACT

Alveologenesis is the final step of lung maturation, which subdivides the alveolar region of the lung into smaller units called alveoli. Each of the nascent dividers serves as a new gas-exchange surface, and collectively they drastically increase the surface area for breathing. Disruption of alveologenesis results in simplification of alveoli, as is seen in premature infants diagnosed with bronchopulmonary dysplasia (BPD), a prevalent lung disease that is often associated with lifelong breathing deficiencies. To date, a majority of studies of alveologenesis rely on two-dimensional (2D) analysis of tissue sections. Given that an overarching theme of alveologenesis is thinning and extension of the epithelium and mesenchyme to facilitate gas exchange, often only a small portion of a cell or a cellular structure is represented in a single 2D plane. Here, we use a three-dimensional (3D) approach to examine the structural architecture and cellular composition of myofibroblasts, alveolar type 2 cells, elastin and lipid droplets in normal as well as BPD-like mouse lung. We found that 2D finger-like septal crests, commonly used to depict growing alveolar septae, are often artifacts of sectioning through fully established alveolar walls. Instead, a more accurate representation of growing septae are 3D ridges that are lined by platelet-derived growth factor receptor alpha (PDGFRA) and alpha smooth muscle actin (α-SMA)-expressing myofibroblasts, as well as the elastin fibers that they produce. Accordingly in 3D, both α-SMA and elastin were each found in connected networks underlying the 3D septal ridges rather than as isolated dots at the tip of 2D septal crests. Analysis through representative stages of alveologenesis revealed unappreciated dynamic changes in these patterns. PDGFRA-expressing cells are only α-SMA-positive during the first phase of alveologenesis, but not in the second phase, suggesting that the two phases of septae formation may be driven by distinct mechanisms. Thin elastin fibers are already present in the alveolar region prior to alveologenesis, suggesting that during alveologenesis, there is not only new elastin deposition, but also extensive remodeling to transform thin and uniformly distributed fibers into thick cables that rim the nascent septae. Analysis of several genetic as well as hyperoxia-induced models of BPD revealed that the myofibroblast organization is perturbed in all, regardless of whether the origin of defect is epithelial, mesenchymal, endothelial or environmental. Finally, analysis of relative position of PDGFRA-positive cells and alveolar type 2 cells reveal that during alveologenesis, these two cell types are not always adjacent to one another. This result suggests that the niche and progenitor relationship afforded by their close juxtaposition in the adult lung may be a later acquired property. These insights revealed by 3D reconstruction of the septae set the foundation for future investigations of the mechanisms driving normal alveologenesis, as well as causes of alveolar simplification in BPD.


Subject(s)
Imaging, Three-Dimensional , Pulmonary Alveoli/growth & development , Actins/metabolism , Animals , Artifacts , Elastin/metabolism , Green Fluorescent Proteins/metabolism , Lipids/chemistry , Mice , Models, Animal , Models, Biological , Myofibroblasts/cytology , Myofibroblasts/metabolism , Pericytes/metabolism , Pulmonary Alveoli/cytology , Receptor, Platelet-Derived Growth Factor alpha/metabolism , Stress, Physiological
14.
Methods Mol Biol ; 1307: 237-43, 2016.
Article in English | MEDLINE | ID: mdl-25403465

ABSTRACT

Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here, we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air-liquid interface for 19-21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology, gene expression, and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and, therefore, offers a promising approach to studying vocal fold biology and biomechanics in health and disease.


Subject(s)
Epithelial Cells/cytology , Human Embryonic Stem Cells/cytology , Mucous Membrane/cytology , Tissue Engineering/methods , Vocal Cords/cytology , Cell Differentiation , Cell Line , Coculture Techniques , Fibroblasts/cytology , Humans , Reproducibility of Results , Tissue Scaffolds
15.
Dev Biol ; 399(2): 263-82, 2015 Mar 15.
Article in English | MEDLINE | ID: mdl-25601450

ABSTRACT

This investigation provides the first systematic determination of the cellular and molecular progression of vocal fold (VF) epithelium development in a murine model. We define five principal developmental events that constitute the progression from VF initiation in the embryonic anterior foregut tube to fully differentiated and functional adult tissue. These developmental events include (1) the initiation of the larynx and vocal folds with apposition of the lateral walls of the primitive laryngopharynx (embryonic (E) day 10.5); (2) the establishment of the epithelial lamina with fusion of the lateral walls of the primitive laryngopharynx (E11.5); (3) the epithelial lamina recanalization and separation of VFs (E13.5-18.5); (4) the stratification of the vocal folds (E13.5-18.5); and (5) the maturation of vocal fold epithelium (postnatal stages). The illustration of these morphogenetic events is substantiated by dynamic changes in cell proliferation and apoptosis, as well as the expression pattern of key transcription factors, FOXA2, SOX2 and NKX2-1 that specify and pattern the foregut endoderm. Furthermore, we documented the gradual conversion of VF epithelial cells from simple precursors expressing cytokeratins 8 and 18 in the embryo into mature stratified epithelial cells also expressing cytokeratins 5 and 14 in the adult. Interestingly, in the adult, cytokeratins 5 and 14 appear to be expressed in all cell layers in the VF, in contrast to their preferential localization to the basal cell layer in surrounding epithelium. To begin investigating the role of signaling molecules in vocal fold development, we characterized the expression pattern of SHH pathway genes, and how loss of Shh affects vocal fold development in the mutant. This study defines the cellular and molecular context and serves as the necessary foundation for future functional investigations of VF formation.


Subject(s)
Gene Expression Regulation, Developmental/physiology , Laryngeal Mucosa/embryology , Morphogenesis/physiology , Vocal Cords/embryology , Animals , Apoptosis/physiology , Cell Proliferation/physiology , Gene Expression Profiling , Gene Expression Regulation, Developmental/genetics , Hepatocyte Nuclear Factor 3-beta/metabolism , Immunohistochemistry , In Situ Hybridization , In Situ Nick-End Labeling , Mice , Nuclear Proteins/metabolism , SOXB1 Transcription Factors/metabolism , Thyroid Nuclear Factor 1 , Transcription Factors/metabolism
16.
Eur J Oral Sci ; 120(6): 495-504, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23167465

ABSTRACT

The MYB family of transcription activators has been associated with a high proliferation rate and an undifferentiated state of cells in a number of tissues. Recently emerging data suggest that these molecules may also play a role in differentiation. In this study, the pattern of expression of c-MYB was followed during postnatal stages of mouse molar odontogenesis using immunohistochemistry on serial sections. Along with an abundance of the c-MYB protein in proliferating zones, we confirmed the presence of this protein in differentiated ameloblasts, odontoblasts, and osteoblasts. In addition, c-MYB was also found in cementoblasts and alveolar fibroblasts. These findings suggest integration of c-MYB into regulatory networks during hard-tissue differentiation and mineralization.


Subject(s)
Alveolar Process/cytology , Cell Differentiation/genetics , Gene Expression Regulation, Developmental/genetics , Genes, myc/genetics , Molar/cytology , Odontogenesis/genetics , Proto-Oncogene Proteins c-myb , Alveolar Process/growth & development , Alveolar Process/metabolism , Ameloblasts/metabolism , Animals , Bone Development/genetics , Bone Development/physiology , Connective Tissue Cells/metabolism , Dental Cementum/metabolism , Gene Expression Regulation, Developmental/physiology , In Situ Nick-End Labeling , Mice , Molar/growth & development , Molar/metabolism , Proto-Oncogene Proteins c-myb/analysis , Proto-Oncogene Proteins c-myb/genetics , Proto-Oncogene Proteins c-myb/metabolism
17.
Eur J Oral Sci ; 119(4): 265-74, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21726286

ABSTRACT

The mouse third molar (M3) develops postnatally and is thus a unique model for studying the integration of a non-mineralized tooth with mineralized bone. This study assessed the morphogenesis of the mouse M3, related to the alveolar bone, comparing M3 development with that of the first molar (M1), the most common model in odontogenesis. The mandibular M3 was evaluated from initiation to eruption by morphology and by assessing patterns of proliferation, apoptosis, osteoclast distribution, and gene expression. Three-dimensional reconstruction and explant cultures were also used. Initiation of M3 occurred perinatally, as an extension of the second molar (M2) which grew into a region of soft mesenchymal tissue above the M2, still far away from the alveolar bone. The bone-free M3 bud gradually became encapsulated by bone at the cap stage at postnatal day 3. Osteoclasts were first visible at postnatal day 4 when the M3 came into close contact with the bone. The number of osteoclasts increased from postnatal day 8 to postnatal day 12 to form a space for the growing tooth. The M3 had erupted by postnatal day 26. The M3, although smaller than the M1, passed through the same developmental stages over a similar time span but showed differences in initiation and in the timing of bone encapsulation.


Subject(s)
Mandible/growth & development , Molar, Third/growth & development , Morphogenesis/physiology , Odontogenesis/physiology , Acid Phosphatase/analysis , Alveolar Process/anatomy & histology , Alveolar Process/growth & development , Animals , Apoptosis/physiology , Biomarkers/analysis , Bone Resorption/pathology , Bone Resorption/physiopathology , Cell Proliferation , Enamel Organ/anatomy & histology , Enamel Organ/growth & development , Fibroblast Growth Factor 4/analysis , Hedgehog Proteins/analysis , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , In Situ Hybridization , Isoenzymes/analysis , Mandible/anatomy & histology , Mice , Molar/anatomy & histology , Molar/growth & development , Molar, Third/anatomy & histology , Osteoblasts/physiology , Osteoclasts/physiology , Osteogenesis/physiology , Proliferating Cell Nuclear Antigen/analysis , Tartrate-Resistant Acid Phosphatase , Tissue Culture Techniques , Tooth Calcification/physiology , Tooth Eruption/physiology , Tooth Germ/anatomy & histology , Tooth Germ/growth & development , Tooth Root/anatomy & histology , Tooth Root/growth & development
18.
Dev Growth Differ ; 53(6): 793-803, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21762405

ABSTRACT

The transcription factor c-Myb is involved in the control of cell proliferation, survival and differentiation. As these processes accompany the morphogenesis of developing teeth, this work investigates the possible role of c-Myb during odontogenesis. Analysis of the expression of c-Myb in the monophyodont mouse was followed by similar analysis in a diphyodont species, the pig, which has a dentition more closely resembling that of the human. The distribution of c-Myb was correlated with the pattern of proliferation and apoptosis and the tooth phenotype of c-Myb mutant mice was also assessed. In the mouse, c-Myb expression was detected throughout prenatal development of the first molar tooth. Negative temporospatial correlation was found between c-Myb expression and apoptosis, while c-Myb expression positively correlated with proliferation. c-Myb-positive cells, however, were more abundant than the proliferating cell nuclear antigen positive cells, suggesting other roles of c-Myb in odontogenesis. In the minipig, in contrast to the mouse, there was an asymmetrical arrangement of c-Myb positive cells, with a higher presence on the labial side of the tooth germ and dental lamina. A cluster of negative cells was situated in the mesenchyme close to the tooth bud. At later stages, the number of positive cells decreased and these cells were situated in the upper part of the dental papilla in the areas of future cusp formation. The expression of c-Myb in both species was strong in the odontoblasts and ameloblasts at the stage of dentin and enamel production suggesting a possible novel role of c-Myb during tooth mineralization.


Subject(s)
Cell Cycle Proteins/metabolism , Odontogenesis , Proto-Oncogene Proteins c-myb/metabolism , Tooth/embryology , Trans-Activators/metabolism , Alleles , Ameloblasts/cytology , Ameloblasts/metabolism , Animals , Apoptosis , Cell Cycle Proteins/genetics , Cell Proliferation , Cloning, Molecular , Dentition , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Immunohistochemistry , In Situ Nick-End Labeling , Mice , Odontoblasts/cytology , Odontoblasts/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Proto-Oncogene Proteins c-myb/genetics , Species Specificity , Swine , Swine, Miniature , Tooth/cytology , Tooth/metabolism , Trans-Activators/genetics
19.
J Anat ; 218(6): 699-716, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21418206

ABSTRACT

The first mouse molar (M1) is the most common model for odontogenesis, with research particularly focused on prenatal development. However, the functional dentition forms postnatally, when the histogenesis and morphogenesis of the tooth is completed, the roots form and the tooth physically anchors into the jaw. In this work, M1 was studied from birth to eruption, assessing morphogenesis, proliferation and apoptosis, and correlating these with remodeling of the surrounding bony tissue. The M1 completed crown formation between postnatal (P) days 0-2, and the development of the tooth root was initiated at P4. From P2 until P12, cell proliferation in the dental epithelium reduced and shifted downward to the apical region of the forming root. In contrast, proliferation was maintained or increased in the mesenchymal cells of the dental follicle. At later stages, before tooth eruption (P20), cell proliferation suddenly ceased. This withdrawal from the cell cycle correlated with tooth mineralization and mesenchymal differentiation. Apoptosis was observed during all stages of M1 postnatal morphogenesis, playing a role in the removal of cells such as osteoblasts in the mandibular region and working together with osteoclasts to remodel the bone around the developing tooth. At more advanced developmental stages, apoptotic cells and bodies accumulated in the cell layers above the tooth cusps, in the path of eruption. Three-dimensional reconstruction of the developing postnatal tooth and bone indicates that the alveolar crypts form by resorption underneath the primordia, whereas the ridges form by active bone growth between the teeth and roots to form a functional complex.


Subject(s)
Molar/growth & development , Odontogenesis , Animals , Apoptosis/physiology , Cell Proliferation , Immunohistochemistry , Mice , Osteoclasts/metabolism , Proliferating Cell Nuclear Antigen/metabolism
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