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.

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.

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.

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.

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.

Isolation of epithelial cells in the developing primary lip and palate.

Failure of the primary lip and palate to fuse leads to clefts of the lip, a birth defect with an incidence of 1 for every 500 in some races. Epithelial cells lining the facial processes of the primary lip and palate, the lateral and medial nasal processes (LNP and MNP), must first make contact to go through a series of highly regulated and coordinated sequence of events to form the normal midface. As yet, many of the basic mechanisms underlying the fusion events of the epithelial-lined surfaces are not known. This is due in part to the difficulty associated with the isolation of the epithelial cells for further study and analysis. The objective of this study was to test the use of laser capture microdissection to collect clean populations of primary lip and palate epithelial cells destined to fuse. Fusing and nonfusing epithelial cell populations of the MNP and LNP were isolated by laser capture microdissection and assayed for gene expression of Bmp-4, Tgfß-2, and their type 1 receptors, Alk-3 and Alk-5, respectively. Transcripts of Bmp-4/Alk-3 and Tgfß-2/Alk-5 were restricted to the epithelial seam of the fusion site, and the epithelium of the prefusion site, in patterns previously reported. Data indicated our ability to isolate clean populations of epithelial and mesenchymal cells around the primary palate fusion site, allowing precise analysis of tissue and site-specific gene expression at high resolution. This study provides the basis of further analysis of the potential molecular players of MNP and LNP fusion and nonfusion of epithelial cells.

Lateral fast muscle fibers originate from the posterior lip of the teleost dermomyotome.

The predominant source of myogenic cells in vertebrates is the dermomyotome (DM). In teleost fish, recent research has provided a useful but limited picture of how myogenic precursors originate from the DM and how they develop into muscle fibers. Here, we combine detailed morphological analysis with examination of molecular markers in trout to describe the cellular mechanisms by which the lateral fast muscle growth zone is created during second phase myogenesis. Results suggest that this occurs by lateral-to-medial immigration of myogenic cells de-epithelializing from the posterior DM lip. These cells then appear to stop proliferation and migrate anteriorly to finally differentiate into muscle fibres. This seems to be a continuation of the rotational cell movement that creates the teleost DM during early somite development. These findings suggest an evolutionary conserved role of the posterior DM lip in amniotes and fish.

Regulation of ectodermal Wnt6 expression by the neural tube is transduced by dermomyotomal Wnt11: a mechanism of dermomyotomal lip sustainment.

Ectodermal Wnt6 plays an important role during development of the somites and the lateral plate mesoderm. In the course of development, Wnt6 expression shows a dynamic pattern. At the level of the segmental plate and the epithelial somites, Wnt6 is expressed in the entire ectoderm overlying the neural tube, the paraxial mesoderm and the lateral plate mesoderm. With somite maturation, expression becomes restricted to the lateral ectoderm covering the ventrolateral lip of the dermomyotome and the lateral plate mesoderm. To study the regulation of Wnt6 expression, we have interfered with neighboring signaling pathways. We show that Wnt1 and Wnt3a signaling from the neural tube inhibit Wnt6 expression in the medial surface ectoderm via dermomyotomal Wnt11. We demonstrate that Wnt11 is an epithelialization factor acting on the medial dermomyotome, and present a model suggesting Wnt11 and Wnt6 as factors maintaining the epithelial nature of the dorsomedial and ventrolateral lips of the dermomyotome, respectively, during dermomyotomal growth.

Differential effects of N-cadherin-mediated adhesion on the development of myotomal waves.

Myotomal fibers form by a first wave of pioneer myoblasts from the medial epithelial somite, and by a second wave from all four lips of the dermomyotome. Then, a third wave of mitotic progenitors colonizes the myotome, initially stemming from the extreme lips and, later, from the central dermomyotome sheet. In vitro studies have suggested that N-cadherin plays a role in myogenesis, but its role in vivo remains poorly understood. We find that during the growth phase of the dermomyotome sheet, when the orientation of mitotic spindles is parallel to the mediolateral extent of the epithelium, N-cadherin protein is inherited by both daughter cells. Prior to dermomyotome dissociation into dermis and muscle progenitors, when mitoses become perpendicularly oriented, N-cadherin remains associated only with the apical cell located in apposition to the myotome, generating molecular asymmetry between basal and apical progeny. Local gene missexpression confirms that N-cadherin-mediated adhesion is sufficient to promote myotome colonization, whereas its absence drives cells towards the subectodermal domain, hence coupling the asymmetric distribution of N-cadherin to a shift in mitotic orientation and to fate segregation. Site-directed electroporation to additional, discrete somite regions, further reveals that N-cadherin-mediated adhesion is necessary for maintaining the epithelial configuration of all dermomyotome domains while promoting the onset of Myod transcription and the translocation into the myotome of myofibers and/or of Pax-positive progenitors. By contrast, N-cadherin has no effect on migration or differentiation of the first wave of myotomal pioneers. Altogether, we show for the first time that the asymmetric localization of N-cadherin during mitosis indirectly influences fate segregation by differentially driving the allocation of progenitors to muscle versus dermal primordia, that the adhesive domain of N-cadherin maintains the integrity of the dermomyotome epithelium, which is necessary for myogenic specification, and that different molecular mechanisms underlie the establishment of pioneer and later myotomal waves.

Epithelial-mesenchymal transformation during craniofacial development.

Epithelial to mesenchymal phenotype transition is a common phenomenon during embryonic development, wound healing, and tumor metastasis. This transition involves cellular changes in cytoskeleton architecture and protein expression. Specifically, this highly regulated biological event plays several important roles during craniofacial development. This review focuses on the regulation of epithelial-mesenchymal transformation (EMT) during neural crest cell migration, and fusion of the secondary palate and the upper lip.

Tissue-plastinated vs. celloidin-embedded large serial sections in video, analog and digital photographic on-screen reproduction: a preliminary step to exact virtual 3D modelling, exemplified in the normal midface and cleft-lip and palate.

This study analyses tissue-plastinated vs. celloidin-embedded large serial sections, their inherent artefacts and aptitude with common video, analog or digital photographic on-screen reproduction. Subsequent virtual 3D microanatomical reconstruction will increase our knowledge of normal and pathological microanatomy for cleft-lip-palate (clp) reconstructive surgery. Of 18 fetal (six clp, 12 control) specimens, six randomized specimens (two clp) were BiodurE12-plastinated, sawn, burnished 90 microm thick transversely (five) or frontally (one), stained with azureII/methylene blue, and counterstained with basic-fuchsin (TP-AMF). Twelve remaining specimens (four clp) were celloidin-embedded, microtome-sectioned 75 microm thick transversely (ten) or frontally (two), and stained with haematoxylin-eosin (CE-HE). Computed-planimetry gauged artefacts, structure differentiation was compared with light microscopy on video, analog and digital photography. Total artefact was 0.9% (TP-AMF) and 2.1% (CE-HE); TP-AMF showed higher colour contrast, gamut and luminance, and CE-HE more red contrast, saturation and hue (P < 0.4). All (100%) structures of interest were light microscopically discerned, 83% on video, 76% on analog photography and 98% in digital photography. Computed image analysis assessed the greatest colour contrast, gamut, luminance and saturation on video; the most detailed, colour-balanced and sharpest images were obtained with digital photography (P < 0.02). TP-AMF retained spatial oversight, covered the entire area of interest and should be combined in different specimens with CE-HE which enables more refined muscle fibre reproduction. Digital photography is preferred for on-screen analysis.

Development of the upper lip.

OBJECTIVES: To affirm and reanalyze George L. Streeter's "merging theory" of upper-lip development in primates by observing progressive embryologic stages in facial development using scanning electron microscopy (SEM) and to further understand upper-lip development. DESIGN: The study was conducted at the California Regional Primate Research Center, Davis. Twenty primate embryos (Macaca fascicularis) and 2 fetuses were examined with SEM. The development of the frontonasal prominence, maxillary prominence, medial nasal prominence, and lateral nasal prominence were sequentially observed. The contribution of these prominences to the formation of the upper lip and nose were carefully analyzed. RESULTS: The maxillary prominence and medial nasal prominence form the upper lip, whereas the lateral nasal, medial nasal, and maxillary prominences form the nose. There is fusion of the maxillary prominence with the medial nasal prominence. This fusion has not been previously described. This has resulted in a modification of the current theory of upper-lip development into one we refer to as the "dynamic fusion theory." CONCLUSIONS: The dynamic fusion theory explains the merging process of the mesenchymal and ecotodermal layers of the facial prominences that contribute to the upper-lip formation. The dynamic fusion theory of facial prominence movement details the interaction between epithelial layers: both epithelial layers must fuse properly to avoid cleft-lip deformities.

Organogenesis particularly relevant to fetal surgery.

In utero surgical intervention is an exciting frontier in medicine. Fetal surgeons strive to treat congenital anomalies definitively while organogenesis is still occurring. Many of these anomalies pose such a threat to the viability of the affected fetus that waiting until after the child is born to treat them is frequently not satisfying and too often unsuccessful. We review the embryology of selected systems that have associated aberrancies of development for which fetal surgery is particularly applicable. The surgeon can more effectively launch an assault against congenital anomalies when armed with a solid appreciation of normal development. Recognizing the critical period for the development of a system allows him or her to formulate the optimal time and mode of intervention.

[Is there new knowledge on embryology and functional anatomy of human mimetic muscles and the upper lip? A contribution to point selection, skin incision and muscle reconstruction in primary lip-nose reconstruction of uni- and bilateral lip-jaw-palatal clefts]. / Gibt es neue Erkenntnisse zur Embryologie und funktionellen Anatomie der humanen mimischen Muskulatur und der Oberlippe? Ein Beitrag zur Punktewahl, Hautschnittführung und Muskelrekonstruktion bei der primären Lippen-Nasen-Plastik ein- und doppelseitiger LKG-Spalten.

BACKGROUND: The great variety of primary cheiloplastic procedures in CLP patients shows that there is disagreement regarding the embryological development of this part of the face, the point selection, skin incision philosophy, and the macroscopic and microscopic functional anatomy of the human muscles of facial expression. We suppose from findings in Asian and African populations that the real embryological development of the upper lip differs from current textbook descriptions. Our own anatomical and embryological investigations serve as a basis for a critical discussion of different techniques of muscle reconstruction, point selection, and skin incision and for a description of an embryologically, functionally, and anatomically oriented operation technique for different entities of CLP. METHODS: The findings of this study result from investigations of the embryonal and early fetal development from the 26th to the 112th i.u. day in REM pictures of the Anatomical Institute of the University of Göttingen (n = 8) and serial histological investigations of the Carnegie and Hooker-Humphrey Collections at the Armed Forces Institute of Pathology, Washington, D.C. (n = 40). Furthermore, we carried out microsurgical dissections of the muscles of facial expression, the osseous and cartilaginous parts of the nose, and the midfacial sutures in two adult heads without congenital disorders and one newborn head with a primary unilateral complete cleft of the lip and alveolus. RESULTS AND DISCUSSION: The formation of the lower third of the upper lip is the result of contact of the maxillary bulges in the midline below the prolabium. According to this finding, the point selections and skin incisions have to be modified in the midline region in different types of uni- and bilateral CLP. Our technique of primary dissection, reorientation, and suturing of the muscles of facial expression is presented. The muscle reconstruction has to be performed independently from the skin preparation.

Epithelial-mesenchymal transformation is the mechanism for fusion of the craniofacial primordia involved in morphogenesis of the chicken lip.

We have previously demonstrated that epithelial-mesenchymal transformation (EMT) brings about TGF beta 3-induced confluence of craniofacial primordia that derive from the maxillary processes and give rise to the avian palate. The upper lip of the chick embryo forms by confluence of primordia also derived from the maxillary processes, but in this case, they fuse with the intermaxillary segment of the nasofrontal process. Here, we ask whether the bilateral epithelial seams formed when these primordia contact each other in vivo are removed by apoptosis (as formerly was believed to occur in developing palate) or by EMT. We found that, as is the case in the palate, the periderm of the two-layered embryonic epithelium begins to slough shortly before these primordia fuse, bringing the basal epithelial cells into close contact. We show by TUNEL staining and confirm by TEM that apoptosis occurs only in periderm. TEM reveals that basal epithelial cells contacting each other to form the midline seam produce numerous desmosomes with each other. Then, basement membrane begins to disappear, numerous filopodia extend from the basal surfaces of epithelial cells, the space between them enlarges, and the seam breaks apart, leaving mesenchymal cells in its wake. Transformation of the carboxyfluorescein (CCFSE)-labeled epithelial seam is demonstrated in vivo by detection of CCFSE bodies in mesenchymal cells that replace it. This demonstration of EMT in avian lip development lays important groundwork for understanding the causes of human cleft lip and analyzing the mechanism of action of growth factors, such as SHH and BMPs, that have been shown (J. A. Helms et al., 1997, Dev. Biol. 187, 25-35) to be involved in avian lip confluence.

Functional matrix cleft repair: a common strategy for unilateral and bilateral clefts.

At first glance, the morphologies of unilateral and bilateral clefts appear quite distinct. So much so, that many authors describe them as separate entities altogether. This has given rise to disparate surgical strategies. This paper presents a unifying theory of pathogenesis and treatment.