Neural crest-derived cells possess differentiation potential to keratinocytes in the process of wound healing.

Neural crest-derived cells (NCDCs), which exist as neural crest cells during the fetal stage and differentiate into palate cells, also exist in adult palate tissues, though with unknown roles. In the present study, NCDCs were labeled with EGFP derived from P0-Cre/CAG-CAT-EGFP (P0-EGFP) double transgenic mice, then their function in palate mucosa wound healing was analyzed. As a palate wound healing model, left-side palate mucosa of P0-EGFP mice was resected, and stem cell markers and keratinocyte markers were detected in healed areas. NCDCs were extracted from normal palate mucosa and precultured with stem cell media for 14 days, then were differentiated into keratinocytes or osteoblasts to analyze pluripotency. The wound healing process started with marginal mucosal regeneration on day two and the entire wound area was lined by regenerated mucosa with EGFP-positive cells (NCDCs) on day 28. EGFP-positive cells comprised approximately 60% of cells in healed oral mucosa, and 65% of those expressed stem cell markers (Sca-1+, PDGFRα+) and 30% expressed a keratinocyte marker (CK13+). In tests of cultured palate mucosa cells, approximately 70% of EGFP-positive cells expressed stem cell markers (Sca-1+, PDGFRα+). Furthermore, under differentiation inducing conditions, cultured EGFP-positive cells were successfully induced to differentiate into keratinocytes and osteoblasts. We concluded that NCDCs exist in adult palate tissues as stem cells and have potential to differentiate into various cell types during the wound healing process.

Functional identification of a rare vascular endothelial growth factor a (VEGFA) variant associating with the nonsyndromic cleft lip with/without cleft palate.

Vascular endothelial growth factor A (VEGFA) is a crucial growth factor, which participates in multiple processes of human growth and development, such as angiogenesis and osteogenesis and is also necessary for development of palate. The purpose of this study was to investigate the effect of a rare VEGFA mutation (NM_001025366.2 773 T > C p.Val258Ala) on the cell functions and osteogenesis. Here, we found that the VEGFA mutation has adverse effects on the function of human embryonic palatal plate mesenchymal (HEPM) cells, and may affect the development of palate. The VEGFA mutation has adverse effects on promoting cell proliferation and migration and inhibiting apoptosis in HEPM and HEK-293 cells. In addition, the mutant VEGFA allele has a negative influence on osteogenesis. Taken together, the rare variant of the VEGFA gene had an adverse effect on cell functions and osteogenesis, which may impact the development of the palate. And these findings suggested that VEGFA mutation (c.773 T > C) may lead to nonsyndromic cleft lip with or without cleft palate and also provide a new insight into the mechanism of VEGFA gene in osteogenesis and palatogenesis.

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds.

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

Anti-epileptic drug topiramate upregulates TGFß1 and SOX9 expression in primary embryonic palatal mesenchyme cells: Implications for teratogenicity.

Topiramate is an anti-epileptic drug that is commonly prescribed not just to prevent seizures but also migraine headaches, with over 8 million prescriptions dispensed annually. Topiramate use during pregnancy has been linked to significantly increased risk of babies born with orofacial clefts (OFCs). However, the exact molecular mechanism of topiramate teratogenicity is unknown. In this study, we first used an unbiased antibody array analysis to test the effect of topiramate on human embryonic palatal mesenchyme (HEPM) cells. This analysis identified 40 differentially expressed proteins, showing strong connectivity to known genes associated with orofacial clefts. However, among known OFC genes, only TGFß1 was significantly upregulated in the antibody array analysis. Next, we validated that topiramate could increase expression of TGFß1 and of downstream target phospho-SMAD2 in primary mouse embryonic palatal mesenchyme (MEPM) cells. Furthermore, we showed that topiramate treatment of primary MEPM cells increased expression of SOX9. SOX9 overexpression in chondrocytes is known to cause cleft palate in mouse. We propose that topiramate mediates upregulation of TGFß1 signaling through activation of γ-aminobutyric acid (GABA) receptors in the palate. TGFß1 and SOX9 play critical roles in orofacial morphogenesis, and their abnormal overexpression provides a plausible etiologic molecular mechanism for the teratogenic effects of topiramate.

T cells participate in bone remodeling during the rapid palatal expansion.

Palatal expansion has been widely used for the treatment of transverse discrepancy or maxillae hypoplasia, but the biological mechanism of bone formation during this procedure is largely unknown. Osteoclasts, which could be regulated by T cells and other components of the immune system, play a crucial role in force-induced bone remodeling. However, whether T cells participate in the palatal expansion process remains to be determined. In this study, we conducted the tooth borne rapid palatal expansion model on the mouse, and detect whether the helper T cells (Th) and regulatory T cells (Treg) could affect osteoclasts and further bone formation. After bonding open spring palatal expanders for 3-day, 5-day, 7-day, and retention for 28-day, micro-computed tomography scanning, histologic, and immunofluorescence staining were conducted to evaluate how osteoclasts were regulated by T cells during the bone remodeling process. We revealed that the increased osteoclast number was downregulated at the end of the early stage of rapid palatal expansion. Type 1 helper T (Th1) cells and Type 17 helper T (Th17) cells increased initially and promoted osteoclastogenesis. Thereafter, the regulatory T (Treg) cells emerged and maintained a relatively high level at the late stage of the experiment to downregulate the osteoclast number by inhibiting Th1 and Th17 cells, which governed the new bone formation. In conclusion, orchestrated T cells are able to regulate osteoclasts at the early stage of rapid palatal expansion and further facilitate bone formation during retention. This study identifies that T cells participate in the palatal expansion procedure by regulating osteoclasts and implies the potential possibility for clinically modulating T cells to improve the palatal expansion efficacy.

Extracellular Matrix Remodeling During Palate Development.

The morphogenesis of the mammalian secondary plate is a series of highly dynamic developmental process, including the palate shelves vertical outgrowth, elevation to the horizontal plane and complete fusion in the midline. Extracellular matrix (ECM) proteins not only form the basic infrastructure for palatal mesenchymal cells to adhere via integrins but also interact with cells to regulate their functions such as proliferation and differentiation. ECM remodeling is essential for palatal outgrowth, expansion, elevation, and fusion. Multiple signaling pathways important for palatogenesis such as FGF, TGF ß, BMP, and SHH remodels ECM dynamics. Dysregulation of ECM such as HA synthesis or ECM breakdown enzymes MMPs or ADAMTS causes cleft palate in mouse models. A better understanding of ECM remodeling will contribute to revealing the pathogenesis of cleft palate.

Comparative gross and scanning electron microscopical study of the oropharyngeal roof of young and adult domestic pigeons (Columba livia domestica).

The present study aims to compare the morphology of the oropharyngeal roof of young and adult domestic pigeon (Columba livia domestica) by gross observation, morphometric measurements, and scanning electron microscopy (SEM). The oropharyngeal roof was divided into the palate and pharyngeal roof. The palate was narrow triangular in shape and concave along its length. It could be divided into a rostral part contained three longitudinal palatine ridges and a caudal part contained the choanal slit. The choanal slit consisted of narrow rostral and wide caudal parts. The edges of the narrow part were encircled by small caudomedially directed papillae. On the contrary, the edges of the wide part of slit were free from papillae. By SEM, the palatal mucosa in young pigeon showed primordia of small papillae which increased in number and size forming a longitudinal row of papillae parallel to the edges of the rostral narrow part of slit in adult pigeon. The surface of the pharyngeal roof appeared smooth in young pigeon, while in adult pigeon, it showed dome-shaped elevations. The infundibular cleft had smooth edges. The caudal part of the pharyngeal roof formed an elevated transverse mucosal fold on which a transverse row of conical-shaped papillae was present. In conclusion, our results documented the presence of some differences between the oropharyngeal roof of young and adult pigeon, which suggest a high degree of functional adaptation in adult pigeon to their diet compared to young pigeon. Such adaptations might increase the efficiency of food prehension in adult pigeon. The present study compared the morphology of the oropharyngeal roof of young and adult domestic pigeon by gross observation, morphometry, and scanning electron microscopy. The morphometrical data showed higher values in adult pigeon compared to young pigeon. The palatal mucosa and the pharyngeal roof of adult pigeon showed papillae and elevations that were not present in young pigeon. Our results suggest a high degree of functional adaptation in adult pigeon to their diet compared to young pigeon. Such adaptations might increase the efficiency of food prehension in adult pigeon.

Oral Soft Tissue Regeneration Using Nano Controlled System Inducing Sequential Release of Trichloroacetic Acid and Epidermal Growth Factor.

BACKGROUND: The effect of nano controlled sequential release of trichloroacetic acid (TCA) and epidermal growth factor (EGF) on the oral soft tissue regeneration was determined. METHODS: Hydrophobically modified glycol chitosan (HGC) nano controlled system was developed for the sequential release of TCA and EGF, and the release pattern was identified. The HGC-based nano controlled release system was injected into the critical-sized defects created in beagles' palatal soft tissues. The palatal impression and its scanned body was obtained on various time points post-injection, and the volumetric amount of soft tissue regeneration was compared among the three groups: CON (natural regeneration control group), EXP1 (TCA-loaded nano controlled release system group), EXP2 (TCA and EGF individually loaded nano controlled release system). DNA microarray analysis was performed and various soft tissue regeneration parameters in histopathological specimens were measured. RESULTS: TCA release was highest at Day 1 whereas EGF release was highest at Day 2 and remained high until Day 3. In the volumetric measurements of impression body scans, no significant difference in soft tissue regeneration between the three groups was shown in two-way ANOVA. However, in the one-way ANOVA at Day 14, EXP2 showed a significant increase in soft tissue regeneration compared to CON. High correlation was determined between the histopathological results of each group. DNA microarray showed up-regulation of various genes and related cell signaling pathways in EXP2 compared to CON. CONCLUSION: HGC-based nano controlled release system for sequential release of TCA and EGF can promote regeneration of oral soft tissue defects.

MicroRNA-21 affects mechanical force-induced midpalatal suture remodelling.

OBJECTIVES: miR-21 can promote osteoblast differentiation of periodontal ligament stem cells. However, the effect of miR-21 on bone remodelling in the midpalatal suture is unclear. This study aimed to elucidate the effects of miR-21 on the midpalatal suture bone remodelling by expanding the palatal sutures. MATERIALS AND METHODS: miR-21 deficient (miR-21-/- ) and wild-type (WT) mice were used to establish animal models by expanding the palatal sutures. Micro-CT, haematoxylin-eosin (HE) staining, tartrate-resistant acid phosphatase (TRAP) staining, fluorescence labelling and immunohistochemistry were used to investigate the function of miR-21 in midpalatal suture bone remodelling. Besides, bone mesenchymal stem cells (BMSCs) derived from both miR-21-/- and WT mice were cultured. The MTT, CCK8, EdU analysis, transwell and wound healing test were used to assess the effects of miR-21 on the characteristics of cells. RESULTS: The expression of ALP was suppressed in miR-21-/- mice after expansion except 28 days. The expression of Ocn in WT mice was much higher than that of miR-21-/-  mice. Besides, with mechanical force, miR-21 deficiency downregulated the expression of Opg, upregulated the expression of Rankl, and induced more osteoclasts as TRAP staining showed. After injecting agomir-21  to miR-21-/- mice, the expression of Alp, Ocn and Opg/Rankl were rescued. In vitro, the experiments suggested that miR-21 deficiency reduced proliferation and migration ability of BMSCs. CONCLUSIONS: The results showed that miR-21 deficiency reduced the rate of bone formation and prolonged the process of bone formation. miR-21 regulated the bone resorption and osteoclastogenesis by affecting the cell abilities of proliferation and migration.

TCDD inhibited the osteogenic differentiation of human fetal palatal mesenchymal cells through AhR and BMP-2/TGF-ß/Smad signaling.

Exposure to environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes cleft palate at high rates, but little is known about the underlying biological mechanisms. In the present study, we cultured osteoblasts from human fetal palate mesenchymal cells (hFPMCs) to explore the effects of TCDD on osteogenic differentiation. The results showed that TCDD significantly decreased cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition. RNA analyses and protein detection demonstrated that TCDD downregulated a wide array of pro-osteogenic biomarkers. Further investigation of the underlying molecular mechanisms revealed that exposure to TCDD activated aryl hydrocarbon receptor (AhR) signaling and inhibited BMP-2/TGF-ß1/Smad pathway molecules. The inactivation of AhR signaling using CRISPR/Cas9-mediated AhR deletion or by genetic siRNA knockdown significantly blocked the effects induced by TCDD, suggesting a critical role of AhR activation in the TCDD-mediated inhibition of hFPMC osteogenic differentiation. The cotreatment with TGF-ß1 or BMP-2 and TCDD significantly relieved the activation of AhR and rescued the impairment of osteogenesis caused by TCDD. Taken together, our findings indicated that TCDD inhibited the osteogenic differentiation of hFPMCs via crosstalk between AhR and BMP-2/TGF-ß1/Smad signaling pathway.

Candida albicans-Cell Interactions Activate Innate Immune Defense in Human Palate Epithelial Primary Cells via Nitric Oxide (NO) and ß-Defensin 2 (hBD-2).

The presence of Candida albicans in the biofilm underlying the dental prosthesis is related to denture stomatitis (DS), an inflammatory reaction of the oral mucosa. The oral epithelium, a component of the innate immune response, has the ability to react to fungal invasion. In this study, we evaluated the in vitro effect of viable C. albicans on the apoptosis, nitric oxide (NO) production, and ß-defensin 2 (hBD-2) expression and production of human palate epithelial cells (HPECs). We further determined whether or not these effects were correlated with fungal invasion of epithelial cells. Interaction between HPEC primary culture and C. albicans was obtained through either direct or indirect cell-cell contact with a supernatant from a hyphal fungus. We found that the hyphae supernatants were sufficient to induce slight HPEC apoptosis, which occurred prior to the activation of the specific mechanisms of epithelial defense. The epithelial defense responses were found to occur via NO and antimicrobial peptide hBD-2 production only during direct contact between C. albicans and HPECs and coincided with the fungus's intraepithelial invasion. However, although the hBD-2 levels remained constant in the HPEC supernatants over time, the NO release and hBD-2 gene expression were reduced at a later time (10 h), indicating that the epithelial defense capacity against the fungal invasion was not maintained in later phases. This aspect of the immune response was associated with increased epithelial invasion and apoptosis maintenance.

Mouse embryonic palatal mesenchymal cells maintain stemness through the PTEN-Akt-mTOR autophagic pathway.

BACKGROUND: Both genetic and environmental factors are implicated in the pathogenesis of cleft palate. However, the molecular and cellular mechanisms that regulate the development of palatal shelves, which are composed of mesenchymal cells, have not yet been fully elucidated. This study aimed to determine the stemness and multilineage differentiation potential of mouse embryonic palatal mesenchyme (MEPM) cells in palatal shelves and to explore the underlying regulatory mechanism associated with cleft palate formation. METHODS: Palatal shelves excised from mice models were cultured in vitro to ascertain whether MEPM are stem cells through immunofluorescence and flow cytometry. The osteogenic, adipogenic, and chondrogenic differentiation potential of MEPM cells were also determined to characterize MEPM stemness. In addition, the role of the PTEN-Akt-mTOR autophagic pathway was investigated using quantitative RT-PCR, Western blotting, and transmission electron microscopy. RESULTS: MEPM cells in culture exhibited cell surface marker expression profiles similar to that of mouse bone marrow stem cells and exhibited positive staining for vimentin (mesodermal marker), nestin (ectodermal marker), PDGFRα, Efnb1, Osr2, and Meox2 (MEPM cells markers). In addition, exposure to PDGFA stimulated chemotaxis of MEPM cells. MEPM cells exhibited stronger potential for osteogenic differentiation as compared to that for adipogenic and chondrogenic differentiation. Undifferentiated MEPM cells displayed a high concentration of autophagosomes, which disappeared after differentiation (at passage four), indicating the involvement of PTEN-Akt-mTOR signaling. CONCLUSIONS: Our findings suggest that MEPM cells are ectomesenchymal stem cells with a strong osteogenic differentiation potential and that maintenance of their stemness via PTEN/AKT/mTOR autophagic signaling prevents cleft palate development.

Embryonic Midfacial Palatal Organ Culture Methods in Developmental Toxicology.

The morphogenesis of the secondary palate provides an interesting model for many of the processes involved in embryonic development. A number of in vitro models have been used to study craniofacial development, including whole embryo culture, palatal mesenchymal and micromass cell cultures, and Trowell-like palatal cultures in which dissected palates are cultured individually or as pairs in contact on a support above medium. This chapter presents a detailed protocol for the culture of maxillary midfacial tissues, including the palatal shelves, in suspension culture. This method involves isolation of the midfacial tissues (maxillary arch and palatal shelves) and suspension of the tissues in medium in flasks. On a rocker in an incubator, the palatal shelves elevate, grow, make contact, and fuse in a time span analogous to that occurring in the intact embryo in utero.

Activity of two hyaluronan preparations on primary human oral fibroblasts.

BACKGROUND AND OBJECTIVE: The potential benefit of using hyaluronan (HA) in reconstructive periodontal surgery is still a matter of debate. The aim of the present study was to evaluate the effects of two HA formulations on human oral fibroblasts involved in soft tissue wound healing/regeneration. MATERIAL AND METHODS: Metabolic, proliferative and migratory abilities of primary human palatal and gingival fibroblasts were examined upon HA treatment. To uncover the mechanisms whereby HA influences cellular behavior, wound healing-related gene expression and activation of signaling kinases were analyzed by qRT-PCR and immunoblotting, respectively. RESULTS: The investigated HA formulations maintained the viability of oral fibroblasts and increased their proliferative and migratory abilities. They enhanced expression of genes encoding type III collagen and transforming growth factor-ß3, characteristic of scarless wound healing. The HAs upregulated the expression of genes encoding pro-proliferative, pro-migratory, and pro-inflammatory factors, with only a moderate effect on the latter in gingival fibroblasts. In palatal but not gingival fibroblasts, an indirect effect of HA on the expression of matrix metalloproteinases 2 and 3 was detected, potentially exerted through induction of pro-inflammatory cytokines. Finally, our data pointed on Akt, Erk1/2 and p38 as the signaling molecules whereby the HAs exert their effects on oral fibroblasts. CONCLUSION: Both investigated HA formulations are biocompatible and enhance the proliferative, migratory and wound healing properties of cell types involved in soft tissue wound healing following regenerative periodontal surgery. Our data further suggest that in gingival tissues, the HAs are not likely to impair the healing process by prolonging inflammation or causing excessive MMP expression at the repair site.

Periderm: Life-cycle and function during orofacial and epidermal development.

Development of the secondary palate involves a complex series of embryonic events which, if disrupted, result in the common congenital anomaly cleft palate. The secondary palate forms from paired palatal shelves which grow initially vertically before elevating to a horizontal position above the tongue and fusing together in the midline via the medial edge epithelia. As the epithelia of the vertical palatal shelves are in contact with the mandibular and lingual epithelia, pathological fusions between the palate and the mandible and/or the tongue must be prevented. This function is mediated by the single cell layered periderm which forms in a distinct and reproducible pattern early in embryogenesis, exhibits highly polarised expression of adhesion complexes, and is shed from the outer surface as the epidermis acquires its barrier function. Disruption of periderm formation and/or function underlies a series of birth defects that exhibit multiple inter-epithelial adhesions including the autosomal dominant popliteal pterygium syndrome and the autosomal recessive cocoon syndrome and Bartsocas Papas syndrome. Genetic analyses of these conditions have shown that IRF6, IKKA, SFN, RIPK4 and GRHL3, all of which are under the transcriptional control of p63, play a key role in periderm formation. Despite these observations, the medial edge epithelia must rapidly acquire the capability to fuse if the palatal shelves are not to remain cleft. This process is driven by TGFß3-mediated, down-regulation of p63 in the medial edge epithelia which allows periderm migration out of the midline epithelial seam and reduces the proliferative potential of the midline epithelial seam thereby preventing cleft palate. Together, these findings indicate that periderm plays a transient but fundamental role during embryogenesis in preventing pathological adhesion between intimately apposed, adhesion-competent epithelia.

Sensory Innervation of the Human Soft Palate.

The human soft palate plays an important role in respiration, swallowing, and speech. These motor activities depend on reflexes mediated by sensory nerve endings. To date, the details of human sensory innervation to the soft palate have not been demonstrated. In this study, eight adult human whole-mount (soft palate-tongue-pharynx-larynx-upper esophagus) specimens were obtained from autopsy. Each specimen was bisected in the midline, forming two equal and symmetrical halves. Eight hemi-specimens were processed with Sihler's stain, a whole-mount nerve staining technique. The remaining eight hemi-soft palates were used for immunohistochemical study. The soft palatal mucosa was dissected from the oral and nasal sides and prepared for neurofilament staining. Our results showed that the sensory nerve fibers formed a dense nerve plexus in the lamina propria of the soft palatal mucosa. There was a significant difference in the innervation density between both sides. Specifically, the oral side had higher density of sensory nerve fibers than the nasal side of the soft palate. The mean number and percent area of the sensory nerve fibers in the mucosa of the nasal side was 78% and 72% of those in the mucosa of the oral side, respectively (P < 0.0001). The data presented here could be helpful for further investigating the morphological and quantitative alterations in the sensory nerves in certain upper airway disorders involving the soft palate such as obstructive sleep apnea (OSA) and for designing effective therapeutic strategies to treat OSA. Anat Rec, 301:1861-1870, 2018. © 2018 Wiley Periodicals, Inc.

Constitutive activation of hedgehog signaling adversely affects epithelial cell fate during palatal fusion.

Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2, to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network.

Somatosensory innervation of the oral mucosa of adult and aging mice.

Oral mechanoreception is implicated in fundamental functions including speech, food intake and swallowing; yet, the neuroanatomical substrates that encode mechanical stimuli are not well understood. Tactile perception is initiated by intricate mechanosensitive machinery involving dedicated cells and neurons. This signal transduction setup is coupled with the topology and mechanical properties of surrounding epithelium, thereby providing a sensitive and accurate system to detect stress fluctuations from the external environment. We mapped the distribution of anatomically distinct neuronal endings in mouse oral cavity using transgenic reporters, molecular markers and quantitative histomorphometry. We found that the tongue is equipped with an array of putative mechanoreceptors that express the principal mechanosensory channel Piezo2, including end bulbs of Krause innervating individual filiform papillae and a novel class of neuronal fibers innervating the epithelium surrounding taste buds. The hard palate and gums are densely populated with three classes of sensory afferents organized in discrete patterns including Merkel cell-neurite complexes, Meissner's corpuscles and glomerular corpuscles. In aged mice, we find that palatal Merkel cells reduce in number at key time-points that correlate with impaired oral abilities, such as swallowing and mastication. Collectively, this work identifies the mechanosensory architecture of oral tissues involved in feeding.

Innervation of the Human Incisive Papilla: Comparison with Other Oral Regions.

Immunohistochemistry for several neurochemical substances was performed on the human incisive papilla and other oral structures. Sodium channel alpha subunit 7 (SCN7A) protein-immunoreactive (IR) Schwann cells and protein gene product 9.5 (PGP 9.5)-IR nerve fibers made nerve plexuses beneath the epithelium of the palate, including the incisive papilla, tongue, and lip. SCN7A immunoreactivity could also be detected in lamellated and nonlamellated capsules of corpuscle endings. Lamellated SCN7A-IR corpuscle endings were mostly restricted to the mucous and cutaneous lips. These endings had thick and spiral-shaped PGP 9.5-IR axons without ramification. Nonlamellated SCN7A-IR corpuscle endings were most numerous in the incisive papilla among the oral regions. On the basis of axonal morphology, the nonlamellated endings were divided into simple and complex types. PGP 9.5-IR terminal axons in the simple type ran straight or meandered with slight ramification, whereas those in the complex type were densely entangled with abundant ramification. Substance P (SP)-, calcitonin gene-related peptide (CGRP)-, and transient receptor potential cation channel subfamily V member 2 (TRPV2)-IR varicose fibers were rarely seen beneath the epithelium of oral structures. The present study indicates that the human incisive papilla has many low-threshold mechanoreceptors with nonlamellated capsules. SP-, CGRP-, and TRPV2-containing nociceptors may be infrequent in the incisive papilla and other oral regions.