Phenytoin Inhibits Cell Proliferation through microRNA-196a-5p in Mouse Lip Mesenchymal Cells.

Cleft lip (CL) is one of the most common birth defects. It is caused by either genetic mutations or environmental factors. Recent studies suggest that environmental factors influence the expression of noncoding RNAs [e.g., microRNA (miRNA)], which can regulate the expression of genes crucial for cellular functions. In this study, we examined which miRNAs are associated with CL. Among 10 candidate miRNAs (miR-98-3p, miR-101a-3p, miR-101b-3p, miR-141-3p, miR-144-3p, miR-181a-5p, miR-196a-5p, miR-196b-5p, miR-200a-3p, and miR-710) identified through our bioinformatic analysis of CL-associated genes, overexpression of miR-181a-5p, miR-196a-5p, miR-196b-5p, and miR-710 inhibited cell proliferation through suppression of genes associated with CL in cultured mouse embryonic lip mesenchymal cells (MELM cells) and O9-1 cells, a mouse cranial neural crest cell line. In addition, we found that phenytoin, an inducer of CL, decreased cell proliferation through miR-196a-5p induction. Notably, treatment with a specific inhibitor for miR-196a-5p restored cell proliferation through normalization of expression of CL-associated genes in the cells treated with phenytoin. Taken together, our results suggest that phenytoin induces CL through miR-196a-5p induction, which suppresses the expression of CL-associated genes.

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.

Msx1 deficiency interacts with hypoxia and induces a morphogenetic regulation during mouse lip development.

Nonsyndromic clefts of the lip and palate are common birth defects resulting from gene-gene and gene-environment interactions. Mutations in human MSX1 have been linked to orofacial clefting and we show here that Msx1 deficiency causes a growth defect of the medial nasal process (Mnp) in mouse embryos. Although this defect alone does not disrupt lip formation, Msx1-deficient embryos develop a cleft lip when the mother is transiently exposed to reduced oxygen levels or to phenytoin, a drug known to cause embryonic hypoxia. In the absence of interacting environmental factors, the Mnp growth defect caused by Msx1 deficiency is modified by a Pax9-dependent 'morphogenetic regulation', which modulates Mnp shape, rescues lip formation and involves a localized abrogation of Bmp4-mediated repression of Pax9 Analyses of GWAS data revealed a genome-wide significant association of a Gene Ontology morphogenesis term (including assigned roles for MSX1, MSX2, PAX9, BMP4 and GREM1) specifically for nonsyndromic cleft lip with cleft palate. Our data indicate that MSX1 mutations could increase the risk for cleft lip formation by interacting with an impaired morphogenetic regulation that adjusts Mnp shape, or through interactions that inhibit Mnp growth.

The molecular anatomy of mammalian upper lip and primary palate fusion at single cell resolution.

The mammalian lip and primary palate form when coordinated growth and morphogenesis bring the nasal and maxillary processes into contact, and the epithelia co-mingle, remodel and clear from the fusion site to allow mesenchyme continuity. Although several genes required for fusion have been identified, an integrated molecular and cellular description of the overall process is lacking. Here, we employ single cell RNA sequencing of the developing mouse face to identify ectodermal, mesenchymal and endothelial populations associated with patterning and fusion of the facial prominences. This analysis indicates that key cell populations at the fusion site exist within the periderm, basal epithelial cells and adjacent mesenchyme. We describe the expression profiles that make each population unique, and the signals that potentially integrate their behaviour. Overall, these data provide a comprehensive high-resolution description of the various cell populations participating in fusion of the lip and primary palate, as well as formation of the nasolacrimal groove, and they furnish a powerful resource for those investigating the molecular genetics of facial development and facial clefting that can be mined for crucial mechanistic information concerning this prevalent human birth defect.

Genetic heterogeneity in Van der Woude syndrome: identification of NOL4 and IRF6 haplotype from the noncoding region as candidates in two families.

Van der Woude syndrome (VWS) shows an autosomal dominant pattern of inheritance with two known candidate genes, IRF6 and GRHL3. In this study, by employing genome-wide linkage analyses on two VWS affected families, we report the cosegregation of an intronic rare variant in NOL4 in one family, and a haplotype consisting of three variants in the noncoding region of IRF6 (introns 1, 8 and 3'UTR) in the other family. Using mouse, as well as human embryos as a model, we demonstrate the expression of NOL4 in the lip and palate primordia during their development. Luciferase, as well as miRNA-transfection assays show decline in the expression of mutant NOL4 construct due to the creation of a binding site for hsa-miR-4796-5p. In family 2, the noncoding region IRF6 haplotype turns out to be the candidate possibly by diminishing its IRF6 expression to half of its normal activity. Thus, here we report a new candidate gene (NOL4) and a haplotype of IRF6 forVWS, and highlight the genetic heterogeneity of this disorder in the Indian population.

Face morphogenesis is promoted by Pbx-dependent EMT via regulation of Snail1 during frontonasal prominence fusion.

Human cleft lip with or without cleft palate (CL/P) is a common craniofacial abnormality caused by impaired fusion of the facial prominences. We have previously reported that, in the mouse embryo, epithelial apoptosis mediates fusion at the seam where the prominences coalesce. Here, we show that apoptosis alone is not sufficient to remove the epithelial layers. We observed morphological changes in the seam epithelia, intermingling of cells of epithelial descent into the mesenchyme and molecular signatures of epithelial-mesenchymal transition (EMT). Utilizing mouse lines with cephalic epithelium-specific Pbx loss exhibiting CL/P, we demonstrate that these cellular behaviors are Pbx dependent, as is the transcriptional regulation of the EMT driver Snail1. Furthermore, in the embryo, the majority of epithelial cells expressing high levels of Snail1 do not undergo apoptosis. Pbx1 loss- and gain-of-function in a tractable epithelial culture system revealed that Pbx1 is both necessary and sufficient for EMT induction. This study establishes that Pbx-dependent EMT programs mediate murine upper lip/primary palate morphogenesis and fusion via regulation of Snail1. Of note, the EMT signatures observed in the embryo are mirrored in the epithelial culture system.

IRF6 and SPRY4 Signaling Interact in Periderm Development.

Rare mutations in IRF6 and GRHL3 cause Van der Woude syndrome, an autosomal dominant orofacial clefting disorder. Common variants in IRF6 and GRHL3 also contribute risk for isolated orofacial clefting. Similarly, variants within genes that encode receptor tyrosine kinase (RTK) signaling components, including members of the FGF pathway, EPHA3 and SPRY2, also contribute risk for isolated orofacial clefting. In the mouse, loss of Irf6 or perturbation of Fgf signaling leads to abnormal oral epithelial adhesions and cleft palate. Oral adhesions can result from a disruption of periderm formation. Here, we find that IRF6 and SPRY4 signaling interact in periderm function. We crossed Irf6 heterozygous ( Irf6+/-) mice with transgenic mice that express Spry4 in the basal epithelial layer ( TgKRT14::Spry4). While embryos with either of these mutations can have abnormal oral adhesions, using a new quantitative assay, we observed a nonadditive effect of abnormal oral epithelial adhesions in the most severely affected double mutant embryos ( Irf6+/-;TgKRT14::Spry4). At the molecular level, the sites of abnormal oral adhesions maintained periderm-like cells that express keratin 6, but we observed abnormal expression of GRHL3. Together, these data suggest that Irf6 and RTK signaling interact in regulating periderm differentiation and function, as well as provide a rationale to screen for epistatic interactions between variants in IRF6 and RTK signaling pathway genes in human orofacial clefting populations.

[Cleft lip and palate]. / Lippen-Kiefer-Gaumen-Spalten.

BACKGROUND: Cleft lip and palate (CLP) represents a group of malformations of unknown etiology but similar phenotypes. This implies consequences for the diagnostics, therapy, prevention, prognosis and risk estimation. OBJECTIVE: Definition of CLP subtypes and the embryonic development, clarification of correlations and differences between entities using epidemiological data, overview of the present state of genetic analyses, correlation to syndromes, sequences and associations and resulting consequences for clinical practice. MATERIAL AND METHODS: Update on embryological development of the face, summary of epidemiological and genetic studies and considerations on pedopathological and forensic aspects. RESULTS: Syndromic and non-syndromic CLP exhibit different and highly variable etiologies, therapeutic needs and prognosis. A thorough understanding is mandatory to distinguish between the different subgroups. In addition to specific aspects of CLP for the pediatric (forensic) pathologist this article provides an overall view of the topic which aims to help understand these malformations.

Sonic hedgehog regulation of Foxf2 promotes cranial neural crest mesenchyme proliferation and is disrupted in cleft lip morphogenesis.

Cleft lip is one of the most common human birth defects, yet our understanding of the mechanisms that regulate lip morphogenesis is limited. Here, we show in mice that sonic hedgehog (Shh)-induced proliferation of cranial neural crest cell (cNCC) mesenchyme is required for upper lip closure. Gene expression profiling revealed a subset of Forkhead box (Fox) genes that are regulated by Shh signaling during lip morphogenesis. During cleft pathogenesis, reduced proliferation in the medial nasal process mesenchyme paralleled the domain of reduced Foxf2 and Gli1 expression. SHH ligand induction of Foxf2 expression was dependent upon Shh pathway effectors in cNCCs, while a functional GLI-binding site was identified downstream of Foxf2 Consistent with the cellular mechanism demonstrated for cleft lip pathogenesis, we found that either SHH ligand addition or FOXF2 overexpression is sufficient to induce cNCC proliferation. Finally, analysis of a large multi-ethnic human population with cleft lip identified clusters of single-nucleotide polymorphisms in FOXF2 These data suggest that direct targeting of Foxf2 by Shh signaling drives cNCC mesenchyme proliferation during upper lip morphogenesis, and that disruption of this sequence results in cleft lip.

The Roles of Hedgehog Signaling in Upper Lip Formation.

Craniofacial development consists of a highly complex sequence of the orchestrated growth and fusion of facial processes. It is also known that craniofacial abnormalities can be detected in 1/3 of all patients with congenital diseases. Within the various craniofacial abnormalities, orofacial clefting is one of the most common phenotypic outcomes associated with retarded facial growth or fusion. Cleft lip is one of the representative and frequently encountered conditions in the spectrum of orofacial clefting. Despite various mechanisms or signaling pathways that have been proposed to be the cause of cleft lip, a detailed mechanism that bridges individual signaling pathways to the cleft lip is still elusive. Shh signaling is indispensable for normal embryonic development, and disruption can result in a wide spectrum of craniofacial disorders, including cleft lip. This review focuses on the current knowledge about the mechanisms of facial development and the etiology of cleft lip that are related to Shh signaling.

MicroRNA expression profiling of the developing murine upper lip.

Clefts of the lip and palate are thought to be caused by genetic and environmental insults but the role of epigenetic mechanisms underlying this common birth defect are unknown. We analyzed the expression of over 600 microRNAs in the murine medial nasal and maxillary processes isolated on GD10.0-GD11.5 to identify those expressed during development of the upper lip and analyzed spatial expression of a subset. A total of 142 microRNAs were differentially expressed across gestation days 10.0-11.5 in the medial nasal processes, and 66 in the maxillary processes of the first branchial arch with 45 common to both. Of the microRNAs exhibiting the largest percent increase in both facial processes were five members of the Let-7 family. Among those with the greatest decrease in expression from GD10.0 to GD11.5 were members of the microRNA-302/367 family that have been implicated in cellular reprogramming. The distribution of expression of microRNA-199a-3p and Let-7i was determined by in situ hybridization and revealed widespread expression in both medial nasal and maxillary facial process, while that for microRNA-203 was much more limited. MicroRNAs are dynamically expressed in the tissues that form the upper lip and several were identified that target mRNAs known to be important for its development, including those that regulate the two main isoforms of p63 (microRNA-203 and microRNA-302/367 family). Integration of these data with corresponding proteomic datasets will lead to a greater appreciation of epigenetic regulation of lip development and provide a better understanding of potential causes of cleft lip.

Nasolabial dimensions of the facial profile at 20 to 37 weeks' gestation on 2- and 3-dimensional sonography in normal Korean fetuses.

OBJECTIVES: The purpose of this study was to evaluate normal nasolabial dimensions using the images of facial profiles in normal Korean fetuses. METHODS: We conducted a cross-sectional study of 355 normal fetuses at 14 to 39 weeks' gestation. After the exclusion of inadequate images and inadequate numbers of fetuses at 14 to 19 and 37 to 39 weeks' gestation, the sonographic facial profiles from 222 fetuses at 20 to 37 weeks' gestation were evaluated. Five parameters, nose length, nose protrusion, pronasal-subnasal distance, distance between the upper philtrum and mouth, and distance between the tip of the nose and mouth, were measured and are presented according to gestational age. Data were analyzed by intraclass correlation coefficients and regression analysis. RESULTS: There were significant linear correlations between gestational age and nose length (R = 0.390; P < .001), pronasal-subnasal distance (R = 0.415; P < .001), and distance between the upper philtrum and mouth (R = 0.315; P < .001). There were significant quadratic relationships between gestational age and nose protrusion (R(2) = 0.213; P < .001) and distance between the tip of the nose and mouth (R(2) = 0.173; P < .001). CONCLUSIONS: We provide preliminary normative nasolabial dimensions of facial profiles at 20 to 37 weeks' gestation in normal Korean fetuses. These data may be of help not only in the understanding of normal nasolabial growth in utero but also in the diagnosis of abnormal facial dimensions.

The mouse as a developmental model for cleft lip and palate research.

Vertebrate and invertebrate model organisms are essential for deciphering biological processes. One of these, the mouse, proved to be a valuable model for understanding the etiopathogenesis of a vast array of human diseases, including congenital malformations such as orofacial clefting conditions. This small mammal's usefulness in cleft lip and palate research stems not only from the striking anatomical and molecular similarities of lip and palate development between human and mouse embryos, but also from its amenability to experimental and genetic manipulation. Using some recent studies as illustrative examples, this review describes different ways of generating and exploiting mouse models to study normal and abnormal development of the lip and palate. Despite a few surmountable disadvantages of using the mouse, numerous mutants have revealed a growing number of molecular key players and have pointed at a tight and complex molecular control during each step of lip and palate development.

Development of the lip and palate: FGF signalling.

The fibroblast growth factor (FGF) signalling pathway is critically involved in several aspects of craniofacial development, including formation of the lip and the palate. FGF receptors are activated by extracellular FGF ligands in order to regulate cellular processes such as migration and morphogenesis through instruction of specific target gene expression. A key factor in the development of orofacial structures is the interaction between mesodermal- and neural crest-derived mesenchyme and ecto- and endodermal-derived epithelium. FGF signalling occurs in both cell types and promotes epithelial-mesenchymal communication through region-specific expression of receptor subtypes. Many FGF ligands and receptors are expressed at specific stages and at precise locations during normal palatogenesis and an absolute requirement of some has been demonstrated by their (conditional) inactivation resulting in a cleft palate phenotype. Other important signalling pathways involving SHH and SPRY are intricately involved in the interpretation of FGF signalling. As a cause of human pathology, functionally validated FGF pathway gene mutations have been exclusively associated with syndromic forms of cleft lip and palate. Most commonly, this includes patients with mutations in FGFR1 and FGFR2 (Kallmann, Pfeiffer, Apert and Crouzon syndromes) where cleft palate is part of a broad craniofacial phenotype, including craniosynostosis. Similarly, FGF8 mutations have been found in patients with Kallmann-like idiopathic hypogonadotropic hypogonadism, some also with cleft lip and palate. In this chapter, we will provide an overview of the relevant FGF ligands and receptors important for lip and palate morphogenesis, correlating their expression patterns with the effects of their perturbation that lead to a clefting pathogenesis.

Roles of BMP signaling pathway in lip and palate development.

Cleft lip with or without cleft palate (CLP) and cleft palate only (CP) are severe disruptions affecting orofacial structures. Patients with orofacial clefts require complex interdisciplinary care, which includes nursing, plastic surgery, maxillofacial surgery, otolaryngology, speech therapy, audiology, psychological and genetic counseling, orthodontics and dental treatment, among others. Overall, treatment of clefts of the lip and palate entails a significant economic burden for families and society. Therefore, prevention is the ultimate objective and this will be facilitated by a complete understanding of the etiology of this condition. Here we review the current concepts regarding the genetic and environmental factors contributing to orofacial clefts and emphasize on the roles of BMP signaling pathway components in the normal and aberrant development of the lip and palate.

Wnt signaling in lip and palate development.

Wnt signaling regulates a variety of cell behaviors and represents a major pathway in development and disease. Mutations in Wnt genes and their downstream targets have been implicated in human craniofacial abnormalities, including the most prevalent birth defect, cleft lip with or without palate. Formation of the upper lip and palate is a complicated process and is composed of a series of highly coordinated steps during tissue morphogenesis, which are rigorously controlled by genetic networks. While genetic controls of lip/palate development have been extensively studied, the roles of Wnt signaling in these processes remained poorly understood. Within the cell, Wnt signaling is transduced in a ß-catenin-dependent (canonical) or -independent (non-canonical) fashion. Recent studies have demonstrated that the canonical and non-canonical pathways play differential roles but both are essential in lip/palate development. Here we review these studies that have substantially advanced our knowledge by elucidating the function of Wnt signaling in upper lip formation, secondary palate development and their disease settings. These advances are important to delineate the genetic networks controlling craniofacial development and to develop personalized therapeutic strategies in related human birth defects in the future.

Divergent regulation of Wnt-mediated development of the dorsomedial and ventrolateral dermomyotomal lips.

The dermomyotome is the dorsal compartment of the somite which gives rise to multiple cell fates including skeletal muscle, connective tissue, and endothelia. It consists of a pseudostratified, roughly rectangular epithelial sheet, the margins of which are called the dermomyotomal lips. The dermomyotomal lips are blastema-like epithelial growth zones, which continuously give rise to resident dermomyotomal cells and emigrating muscle precursor cells, which populate the subjacent myotomal compartment. Wnt signaling has been shown to regulate both dermomyotome formation and maintenance of the dermomyotomal lips. Whereas the epithelialization of the dermomyotome is regulated via canonical, ß-catenin-dependent Wnt signaling, the downstream signaling mechanisms suppressing epithelial-mesenchymal transition (EMT) in the mature dermomyotomal lips have been unknown. Here, we present evidence that dermomyotomal lip sustainment is differentially regulated. Whereas the dorsomedial dermomyotomal lip is maintained by canonical Wnt signaling, development of the ventrolateral dermomyotomal lip is regulated by non-canonical, PCP-like Wnt signaling. We discuss our results in the light of the different developmental prerequisites in the dorsomedial and ventrolateral lips, respectively, thus providing a new perspective on the regulation of dermomyotomal EMT.

Effects of embryonic hypoxia on lip formation.

The upper lip is formed by the fusion of facial processes, a process in which many genetic and environmental factors are involved. Embryonic hypoxia is induced by uterine anemia and the administration of vasoconstrictors during pregnancy. To define the relationship between hypoxia and upper lip formation, hypoxic conditions were created in a whole embryo culture system. Hypoxic embryos showed a high frequency of impaired fusion, reflecting failure in the growth of the lateral nasal process (LNP). In hypoxic embryos, cell proliferation activity in the LNP mesenchyme was decreased following downregulation of genes that are involved in lip formation. We also observed upregulation of vascular endothelial growth factor expression along with the induction of apoptosis in the LNP. These results suggest that embryonic hypoxia during lip formation induces apoptosis in physiologically hypoxic regions, hypoxia-induced gene expression and downregulation of the genes involved in maxillofacial morphogenesis as immediate responses, followed by reduction of mesenchymal cell proliferation activity, resulting in insufficient growth of the facial processes.

Maintenance of certification: unilateral cleft lip repair.

Cleft lip repair is arguably one of the defining procedures in the field of plastic surgery. The results may be dramatic both visually and emotionally. The variety of techniques of cleft lip repair is well described in the literature, in part because of its inherent difficulty to master. The following article reviews the history of cleft lip repair and attempts to make clear the techniques of the rotation-advancement-type repair.