Functions of Matricellular Proteins in Dental Tissues and Their Emerging Roles in Orofacial Tissue Development, Maintenance, and Disease.

Matricellular proteins (MCPs) are defined as extracellular matrix (ECM) associated proteins that are important regulators and integrators of microenvironmental signals, contributing to the dynamic nature of ECM signalling. There is a growing understanding of the role of matricellular proteins in cellular processes governing tissue development as well as in disease pathogenesis. In this review, the expression and functions of different MP family members (periostin, CCNs, TSPs, SIBLINGs and others) are presented, specifically in relation to craniofacial development and the maintenance of orofacial tissues, including bone, gingiva, oral mucosa, palate and the dental pulp. As will be discussed, each MP family member has been shown to have non-redundant roles in development, tissue homeostasis, wound healing, pathology and tumorigenesis of orofacial and dental tissues.

Mouth opening is mediated by separation of dorsal and ventral daughter cells of the lip precursor cells in the larvacean, Oikopleura dioica.

Mouth formation involves the processes of mouth opening, formation of the oral cavity, and the development of associated sensory organs. In deuterostomes, the surface ectoderm and the anterior part of the archenteron are reconfigured and reconnected to make a mouth opening. This study of the larval development of the larvacean, Oikopleura dioica, investigates the cellular organization of the oral region, the developmental processes of the mouth, and the formation of associated sensory cells. O. dioica is a simple chordate whose larvae are transparent and have a small number of constituent cells. It completes organ morphogenesis in 7 h, between hatching 3 h after fertilization and the juvenile stage at 10 h, when it attains adult form and starts to feed. It has two types of mechanosensory cell embedded in the oral epithelium, which is a single layer of cells. There are twenty coronal sensory cells in the circumoral nerve ring and two dorsal sensory organ cells. Two bilateral lip precursor cells (LPCs), facing the anterior surface, divide dorsoventrally and make a wedge-shaped cleft between the two daughter cells named the dorsal lip cell (DLC) and the ventral lip cell (VLC). Eventually, the DLC and VLC become detached and separated into dorsal and ventral lips, triggering mouth opening. This is an intriguing example of cell division itself contributing to morphogenesis. The boundary between the ectoderm and endoderm is present between the lip cells and coronal sensory cells. All oral sensory cells, including dorsal sensory organ cells, were of endodermal origin and were not derived from the ectodermal placode. These observations on mouth formation provide a cellular basis for further studies at a molecular level, in this simple chordate.

Nucleic acid methylation and orofacial morphogenesis.

In this review, we highlight the current state of knowledge of the diverse roles nucleic acid methylation plays in the embryonic development of the orofacial region and how aberrant methylation may contribute to orofacial clefts. We also consider the role of methylation in the regulation of neural crest cell function as it pertains to orofacial ontogeny. Changes in DNA methylation, as a consequence of environmental effects, have been observed in the regulatory regions of several genes, potentially identifying new candidate genes for orofacial clefting and opening promising new avenues for further research. While the focus of this review is primarily on the nonsyndromic forms of orofacial clefting, syndromic forms are briefly discussed in the context of aberrant nucleic acid methylation.

RDH10 function is necessary for spontaneous fetal mouth movement that facilitates palate shelf elevation.

Cleft palate is a common birth defect, occurring in approximately 1 in 1000 live births worldwide. Known etiological mechanisms of cleft palate include defects within developing palate shelf tissues, defects in mandibular growth and defects in spontaneous fetal mouth movement. Until now, experimental studies directly documenting fetal mouth immobility as an underlying cause of cleft palate have been limited to models lacking neurotransmission. This study extends the range of anomalies directly demonstrated to have fetal mouth movement defects correlated with cleft palate. Here, we show that mouse embryos deficient in retinoic acid (RA) have mispatterned pharyngeal nerves and skeletal elements that block spontaneous fetal mouth movement in utero Using X-ray microtomography, in utero ultrasound video, ex vivo culture and tissue staining, we demonstrate that proper retinoid signaling and pharyngeal patterning are crucial for the fetal mouth movement needed for palate formation. Embryos with deficient retinoid signaling were generated by stage-specific inactivation of retinol dehydrogenase 10 (Rdh10), a gene crucial for the production of RA during embryogenesis. The finding that cleft palate in retinoid deficiency results from a lack of fetal mouth movement might help elucidate cleft palate etiology and improve early diagnosis in human disorders involving defects of pharyngeal development.

Pierre Robin Sequence.

Pierre Robin sequence consists of clinical triad of micrognathia, glossoptosis, and airway compromise with variable inclusion of cleft palate. Evaluation of airway obstruction includes physical examination, polysomnography for obstruction events, and a combination of nasoendoscopy and bronchoscopy to search for synchronous obstructive lesions. A multidisciplinary approach is required given the high rate of syndromic disease. Management of airway obstruction and feeding starts with nonsurgical maneuvers, such as prone and lateral positioning, nasopharyngeal stenting, and continuous positive airway pressure. Surgical management includes mandibular distraction and tongue-lip adhesion. Subglottic obstruction and central sleep apnea may best be treated with tracheostomy.

Roles of FGF8 subfamily in embryogenesis and oral­maxillofacial diseases (Review).

Fibroblast growth factors (FGFs) are diffusible polypeptides released by a variety of cell types. FGF8 subfamily members regulate embryonic development processes through controlling progenitor cell growth and differentiation, and are also functional in adults in tissue repair to maintain tissue homeostasis. FGF8 family members exhibit unique binding affinities with FGF receptors and tissue distribution patterns. Increasing evidence suggests that, by regulating multiple cellular signaling pathways, alterations in the FGF8 subfamily are involved in craniofacial development, odontogenesis, tongue development and salivary gland branching morphogenesis. Aberrant FGF signaling transduction, caused by mutations as well as abnormal expression or isoform splicing, plays an important role in the development of oral diseases. Targeting FGF8 subfamily members provides a new promising strategy for the treatment of oral diseases. The aim of this review was to summarize the aberrant regulations of FGF8 subfamily members and their potential implications in oral­maxillofacial diseases.

Two independent sulfation processes regulate mouth-form plasticity in the nematode Pristionchus pacificus.

Sulfation of biomolecules, like phosphorylation, is one of the most fundamental and ubiquitous biochemical modifications with important functions during detoxification. This process is reversible, involving two enzyme classes: a sulfotransferase, which adds a sulfo group to a substrate; and a sulfatase that removes the sulfo group. However, unlike phosphorylation, the role of sulfation in organismal development is poorly understood. In this study, we find that two independent sulfation events regulate the development of mouth morphology in the nematode Pristionchus pacificus. This nematode has the ability to form two alternative mouth morphologies depending on environmental cues, an example of phenotypic plasticity. We found that, in addition to a previously described sulfatase, a sulfotransferase is involved in regulating the mouth-form dimorphism in P. pacificus However, it is unlikely that both of these sulfation-associated enzymes act upon the same substrates, as they are expressed in different cell types. Furthermore, animals mutant in genes encoding both enzymes show condition-dependent epistatic interactions. Thus, our study highlights the role of sulfation-associated enzymes in phenotypic plasticity of mouth structures in Pristionchus.

The development of a core syllabus for the teaching of oral anatomy, histology, and embryology to dental students via an international 'Delphi Panel'.

The formulation of core syllabuses for the biomedical sciences within medical and dental courses is partially driven by the need to cope with decreased time allocations for these subjects as a result of major curricular changes taking place worldwide. There is also a requirement to deal with the request for increased clinical relevance. In response to such demands, the International Federation of Associations of Anatomists (IFAA) is devising core syllabuses for the anatomical sciences relating to the education and training of both scientific and clinical professions. The process initially involves using Delphi Panels consisting of a team of anatomists, scientists, and clinicians who evaluate syllabus content in detail and accord each element/topic 'essential,' 'important,' 'acceptable,' or 'not required' status. Their conjectures, published on the IFAA website, provide merely a framework to enable other stakeholders to comment. The approach is international in scope, is conceptually 'democratic,' and is developmentally fluid in being readily available for amendment. The aim is to set internationally recognized standards and thus to provide guidelines concerning anatomical knowledge when engaged in course development. This article presents the deliberations of an IFAA Delphi Panel into a core syllabus for oral anatomy, histology, and embryology within the dental curriculum. Clin. Anat. 31:231-249, 2018. © 2017 Wiley Periodicals, Inc.

Hedgehog participates in the establishment of left-right asymmetry during amphioxus development by controlling Cerberus expression.

Correct patterning of left-right (LR) asymmetry is essential during the embryonic development of bilaterians. Hedgehog (Hh) signaling is known to play a role in LR asymmetry development of mouse, chicken and sea urchin embryos by regulating Nodal expression. In this study, we report a novel regulatory mechanism for Hh in LR asymmetry development of amphioxus embryos. Our results revealed that Hh-/- embryos abolish Cerberus (Cer) transcription, with bilaterally symmetric expression of Nodal, Lefty and Pitx In consequence, Hh-/- mutants duplicated left-side structures and lost right-side characters, displaying an abnormal bilaterally symmetric body plan. These LR defects in morphology and gene expression could be rescued by Hh mRNA injection. Our results indicate that Hh participates in amphioxus LR patterning by controlling Cer gene expression. Curiously, however, upregulation of Hh signaling failed to alter the Cer expression pattern or LR morphology in amphioxus embryos, indicating that Hh might not provide an asymmetric cue for Cer expression. In addition, Hh is required for mouth opening in amphioxus, hinting at a homologous relationship between amphioxus and vertebrate mouth development.

The Dlx5-FGF10 signaling cascade controls cranial neural crest and myoblast interaction during oropharyngeal patterning and development.

Craniofacial development depends on cell-cell interactions, coordinated cellular movement and differentiation under the control of regulatory gene networks, which include the distal-less (Dlx) gene family. However, the functional significance of Dlx5 in patterning the oropharyngeal region has remained unknown. Here, we show that loss of Dlx5 leads to a shortened soft palate and an absence of the levator veli palatini, palatopharyngeus and palatoglossus muscles that are derived from the 4th pharyngeal arch (PA); however, the tensor veli palatini, derived from the 1st PA, is unaffected. Dlx5-positive cranial neural crest (CNC) cells are in direct contact with myoblasts derived from the pharyngeal mesoderm, and Dlx5 disruption leads to altered proliferation and apoptosis of CNC and muscle progenitor cells. Moreover, the FGF10 pathway is downregulated in Dlx5-/- mice, and activation of FGF10 signaling rescues CNC cell proliferation and myogenic differentiation in these mutant mice. Collectively, our results indicate that Dlx5 plays crucial roles in the patterning of the oropharyngeal region and development of muscles derived from the 4th PA mesoderm in the soft palate, likely via interactions between CNC-derived and myogenic progenitor cells.

Nitric Oxide regulates mouth development in amphioxus.

The development of the mouth in animals has fascinated researchers for decades, and a recent study proposed the modern view of recurrent evolution of protostomy and deuterostomy. Here we expanded our knowledge about conserved traits of mouth formation in chordates, testing the hypothesis that nitric oxide (NO) is a potential regulator of this process. In the present work we show for the first time that NO is an essential cell signaling molecule for cephalochordate mouth formation, as previously shown for vertebrates, indicating its conserved ancestral role in chordates. The experimental decrease of NO during early amphioxus Branchiostoma lanceolatum development impaired the formation of the mouth and gill slits, demonstrating that it is a prerequisite in pharyngeal morphogenesis. Our results represent the first step in the understanding of NO physiology in non-vertebrate chordates, opening new evolutionary perspectives into the ancestral importance of NO homeostasis and acquisition of novel biological roles during evolution.

Pre-oral gut contributes to facial structures in non-teleost fishes.

Despite the wide variety of adaptive modifications in the oral and facial regions of vertebrates, their early oropharyngeal development is considered strictly uniform. It involves sequential formation of the mouth and pharyngeal pouches, with ectoderm outlining the outer surface and endoderm the inner surface, as a rule. At the extreme anterior domain of vertebrate embryos, the ectoderm and endoderm directly juxtapose and initial development of this earliest ecto-endoderm interface, the primary mouth, typically involves ectodermal stomodeal invagination that limits the anterior expansion of the foregut endoderm. Here we present evidence that in embryos of extant non-teleost fishes, oral (stomodeal) formation is preceded by the development of prominent pre-oral gut diverticula (POGD) between the forebrain and roof of the forming mouth. Micro-computed tomography (micro-CT) imaging of bichir, sturgeon and gar embryos revealed that foregut outpocketing at the pre-oral domain begins even before the sequential formation of pharyngeal pouches. The presence of foregut-derived cells in the front of the mouth was further confirmed by in vivo experiments that allowed specific tracing of the early endodermal lining. We show that POGD in sturgeons contribute to the orofacial surface of their larvae, comprising oral teeth, lips, and sensory barbels. To our knowledge, this is the first thorough evidence for endodermal origin of external craniofacial structures in any vertebrate. In bichir and gar embryos, POGD form prominent cranial adhesive organs that are characteristic of the ancient bauplan of free-living chordate larvae. POGD hence seem arguably to be ancestral for all ray-finned fishes, and their topology, pharyngeal-like morphogenesis and gene expression suggest that they are evolutionarily related to the foregut-derived diverticula of early chordate and hemichordate embryos. The formation of POGD might thus represent an ancestral developmental module with deep deuterostome origins.

Conservation of BMP2/4 expression patterns within the clade Branchiostoma (amphioxus): Resolving interspecific discrepancies.

In 2016, Kaji et al. concluded that the amphioxus mouth has the quality of a coelomoduct and is, therefore, not homologous to the oral opening of any other animal. They studied a Japanese population of Branchiostoma japonicum and based their conclusion, in part, on the larval expression of BMP2/4 in cells that reportedly joined the rim of the forming mouth. They did not detect transcription of that gene in any other tissues in the anterior region of the larva. Their results were almost the inverse of findings for B. floridae by Panopoulou et al. (1998), who detected BMP2/4 expression in several anterior tissues, but not in cells intimately associated with the nascent mouth. To resolve this discrepancy, we have studied BMP2/4 in a Chinese population of B. japonicum as well as in an additional species, the European B. lanceolatum. In both species, larval expression of BMP2/4 closely resembles the pattern previously reported for B. floridae-that is, transcription is undetectable in tissues juxtaposed to the forming mouth, but is seen in several other anterior structures (most conspicuously in the lining of the rostral coelom and the club-shaped gland). In sum, we could not repeat the BMP2/4 expression pattern of Kaji et al. (2016) even in the same species, and their findings for this gene, at least, cannot be counted as a support for their hypothesis for a coelomoduct mouth.

Mouth development.

A mouth is present in all animals, and comprises an opening from the outside into the oral cavity and the beginnings of the digestive tract to allow eating. This review focuses on the earliest steps in mouth formation. In the first half, we conclude that the mouth arose once during evolution. In all animals, the mouth forms from ectoderm and endoderm. A direct association of oral ectoderm and digestive endoderm is present even in triploblastic animals, and in chordates, this region is known as the extreme anterior domain (EAD). Further support for a single origin of the mouth is a conserved set of genes that form a 'mouth gene program' including foxA and otx2. In the second half of this review, we discuss steps involved in vertebrate mouth formation, using the frog Xenopus as a model. The vertebrate mouth derives from oral ectoderm from the anterior neural ridge, pharyngeal endoderm and cranial neural crest (NC). Vertebrates form a mouth by breaking through the body covering in a precise sequence including specification of EAD ectoderm and endoderm as well as NC, formation of a 'pre-mouth array,' basement membrane dissolution, stomodeum formation, and buccopharyngeal membrane perforation. In Xenopus, the EAD is also a craniofacial organizer that guides NC, while reciprocally, the NC signals to the EAD to elicit its morphogenesis into a pre-mouth array. Human mouth anomalies are prevalent and are affected by genetic and environmental factors, with understanding guided in part by use of animal models. WIREs Dev Biol 2017, 6:e275. doi: 10.1002/wdev.275 For further resources related to this article, please visit the WIREs website.

Evolutionary aspects of the development of teeth and baleen in the bowhead whale.

In utero, baleen whales initiate the development of several dozens of teeth in upper and lower jaws. These tooth germs reach the bell stage and are sometimes mineralized, but toward the end of prenatal life they are resorbed and no trace remains after birth. Around the time that the germs disappear, the keratinous baleen plates start to form in the upper jaw, and these form the food-collecting mechanism. Baleen whale ancestors had two generations of teeth and never developed baleen, and the prenatal teeth of modern fetuses are usually interpreted as an evolutionary leftover. We investigated the development of teeth and baleen in bowhead whale fetuses using histological and immunohistochemical evidence. We found that upper and lower dentition initially follow similar developmental pathways. As development proceeds, upper and lower tooth germs diverge developmentally. Lower tooth germs differ along the length of the jaw, reminiscent of a heterodont dentition of cetacean ancestors, and lingual processes of the dental lamina represent initiation of tooth bud formation of replacement teeth. Upper tooth germs remain homodont and there is no evidence of a secondary dentition. After these germs disappear, the oral epithelium thickens to form the baleen plates, and the protein FGF-4 displays a signaling pattern reminiscent of baleen plates. In laboratory mammals, FGF-4 is not involved in the formation of hair or palatal rugae, but it is involved in tooth development. This leads us to propose that the signaling cascade that forms teeth in most mammals has been exapted to be involved in baleen plate ontogeny in mysticetes.

Role of JNK during buccopharyngeal membrane perforation, the last step of embryonic mouth formation.

BACKGROUND: The buccopharyngeal membrane is a thin layer of cells covering the embryonic mouth. The perforation of this structure creates an opening connecting the external and the digestive tube which is essential for oral cavity formation. In humans, persistence of the buccopharyngeal membrane can lead to orofacial defects such as choanal atresia, oral synechiaes, and cleft palate. Little is known about the causes of a persistent buccopharyngeal membrane and, importantly, how this structure ruptures. RESULTS: We have determined, using antisense and pharmacological approaches, that Xenopus embryos deficient c-Jun N-terminal kinase (JNK) signaling have a persistent buccopharyngeal membrane. JNK deficient embryos have decreased cell division and increased cellular stress and apoptosis. However, altering these processes independently of JNK did not affect buccopharyngeal membrane perforation. JNK deficient embryos also have increased intercellular adhesion and defects in e-cadherin localization. Conversely, embryos with overactive JNK have epidermal fragility, increased E-cadherin internalization, and increased membrane localized clathrin. In the buccopharyngeal membrane, clathrin is colocalized with active JNK. Furthermore, inhibition of endocytosis results in a persistent buccopharyngeal membrane, mimicking the JNK deficient phenotype. CONCLUSIONS: The results of this study suggest that JNK has a role in the disassembly adherens junctions by means of endocytosis that is required during buccopharyngeal membrane perforation. Developmental Dynamics 246:100-115, 2017. © 2016 Wiley Periodicals, Inc.

Exploring the potential of laser capture microdissection technology in integrated oral biosciences.

Laser capture microdissection (LCM) is a high-end research and diagnostic technology that helps in obtaining pure cell populations for the purpose of cell- or lesion-specific genomic and proteomic analysis. Literature search on the application of LCM in oral tissues was made through PubMed. There is ample evidence to substantiate the utility of LCM in understanding the underlying molecular mechanism involving an array of oral physiological and pathological processes, including odontogenesis, taste perception, eruptive tooth movement, oral microbes, and cancers of the mouth and jaw tumors. This review is aimed at exploring the potential application of LCM in oral tissues as a high-throughput tool for integrated oral sciences. The indispensable application of LCM in the construction of lesion-specific genomic libraries with emphasis on some of the novel molecular markers thus discovered is also highlighted.

Interpreting amphioxus, and thoughts on ancestral chordate mouths and brains.

Amphioxus is increasingly important as a model for ancestral chordates. Nevertheless, it is secondarily modified in various ways, especially in the larva, whose small size has resulted in a rescaling and repositioning of structures. This is especially pronounced in the head region, where the mouth opens asymmetrically on the left side, leading to speculation that the mouth is secondarily derived, e.g. from a gill slit, and is hence not homologous with mouths in other animals. The available evidence does not, in the author's view, support this interpretation. A second issue is raised concerning the identity and function of the midbrain homolog, whose extent depends on whether greater weight is given to dorsal landmarks in the nerve cord or ventral ones. The presence of two sets of dorsal photoreceptors, the lamellar body and Joseph cells, functionally links the region they occupy to the vertebrate midbrain. The midbrain is currently suggested to be the brain region in which primary consciousness emerged during early vertebrate evolution, so the origin of its constituent cells is of special interest. Possible amphioxus homologs include the anterior-most group of dorsal bipolar cells (ADBs), which are apico-basally inverted (i.e. synapse-bearing neurites arise from the apical cell compartment) in the same fashion as cortical neurons in vertebrates. This may have been a crucial innovation for chordates, responsible for both improved sensory processing and, eventually, consciousness.

One Odontogenic Cell-Population Contributes to the Development of the Mouse Incisors and of the Oral Vestibule.

The area of the oral vestibule is often a place where pathologies appear (e.g., peripheral odontomas). The origin of these pathologies is not fully understood. In the present study, we traced a cell population expressing Sonic hedgehog (Shh) from the beginning of tooth development using Cre-LoxP system in the lower jaw of wild-type (WT) mice. We focused on Shh expression in the area of the early appearing rudimentary incisor germs located anteriorly to the prospective incisors. The localization of the labelled cells in the incisor germs and also in the inner epithelial layer of the vestibular anlage showed that the first very early developmental events in the lower incisor area are common to the vestibulum oris and the prospective incisor primordia in mice. Scanning electron microscopic analysis of human historical tooth-like structures found in the vestibular area of jaws confirmed their relation to teeth and thus the capability of the vestibular tissue to form teeth. The location of labelled cells descendant of the early appearing Shh expression domain related to the rudimentary incisor anlage not only in the rudimentary and functional incisor germs but also in the externally located anlage of the oral vestibule documented the odontogenic potential of the vestibular epithelium. This potential can be awakened under pathological conditions and become a source of pathologies in the vestibular area.