Palatal Fusion Research Articles

Spatial Transcriptomics Unravel the Tissue Complexity of Oral Pathogenesis.

Spatial transcriptomics (ST) is a cutting-edge methodology that enables the simultaneous profiling of global gene expression and spatial information within histological tissue sections. Traditional transcriptomic methods lack the spatial resolution required to sufficiently examine the complex interrelationships between cellular regions in diseased and healthy tissue states. We review the general workflows for ST, from specimen processing to ST data analysis and interpretations of the ST dataset using visualizations and cell deconvolution approaches. We show how recent studies used ST to explore the development or pathogenesis of specific craniofacial regions, including the cranium, palate, salivary glands, tongue, floor of mouth, oropharynx, and periodontium. Analyses of cranial suture patency and palatal fusion during development using ST identified spatial patterns of bone morphogenetic protein in sutures and osteogenic differentiation pathways in the palate, in addition to the discovery of several genes expressed at critical locations during craniofacial development. ST of salivary glands from patients with Sjögren's disease revealed co-localization of autoimmune antigens with ductal cells and a subpopulation of acinar cells that was specifically depleted by the dysregulated autoimmune response. ST of head and neck lesions, such as premalignant leukoplakia progressing to established oral squamous cell carcinomas, oral cancers with perineural invasions, and oropharyngeal lesions associated with HPV infection spatially profiled the complex tumor microenvironment, showing functionally important gene signatures of tumor cell differentiation, invasion, and nontumor cell dysregulation within patient biopsies. ST also enabled the localization of periodontal disease-associated gene expression signatures within gingival tissues, including genes involved in inflammation, and the discovery of a fibroblast subtype mediating the transition between innate and adaptive immune responses in periodontitis. The increased use of ST, especially in conjunction with single-cell analyses, promises to improve our understandings of craniofacial development and pathogenesis at unprecedented tissue-level resolution in both space and time.

Single-cell transcriptome and chromatin accessibility mapping of upper lip and primary palate fusion.

Cleft lip and/or primary palate (CL/P) represent a prevalent congenital malformation, the aetiology of which is highly intricate. Although it is generally accepted that the condition arises from failed fusion between the upper lip and primary palate, the precise mechanism underlying this fusion process remains enigmatic. In this study, we utilized transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) to interrogate lambdoidal junction tissue derived from C57BL/6J mouse embryos at critical stages of embryogenesis (10.5, 11.5 and 12.5 embryonic days). We successfully identified distinct subgroups of mesenchymal and ectodermal cells involved in the fusion process and characterized their unique transcriptional profiles. Furthermore, we conducted cell differentiation trajectory analysis, revealing a dynamic repertoire of genes that are sequentially activated or repressed during pseudotime, facilitating the transition of relevant cell types. Additionally, we employed scATAC data to identify key genes associated with the fusion process and demonstrated differential chromatin accessibility across major cell types. Finally, we constructed a dynamic intercellular communication network and predicted upstream transcriptional regulators of critical genes involved in important signalling pathways. Our findings provide a valuable resource for future studies on upper lip and primary palate development, as well as congenital defects.

Palatal segment contributions to midfacial anterior-posterior growth.

Anterior-posterior (A-P) elongation of the palate is a critical aspect of integrated midfacial morphogenesis. Reciprocal epithelial-mesenchymal interactions drive secondary palate elongation that is coupled to the periodic formation of signaling centers within the rugae growth zone (RGZ). However, the relationship between RGZ driven morphogenetic processes, the differentiative dynamics of underlying palatal bone mesenchymal precursors, and the segmental organization of the upper jaw has remained enigmatic. A detailed ontogenetic study of these relationships is important, because palatal segment growth is a critical aspect of normal midfacial growth, can be modified to produce dysmorphology, and is a likely basis for evolutionary differences in upper jaw morphology. Variation in palatal-segment specific growth may also underlie known differences in palatal segment proportions between inbred mouse strains. We completed a combined whole mount gene expression and morphometric analysis of normal murine palatal growth dynamics and their association with palatal segment elongation and resulting upper jaw morphology. Our results demonstrated that the first formed palatal ruga (ruga 1), found just posterior to the RGZ, maintained an association with important nasal, neurovascular and palatal structures throughout early midfacial development; suggesting that these features are positioned at a proximal source of embryonic midfacial directional growth. Our detailed characterization of midfacial morphogenesis revealed a one-to-one relationship between palatal segments and upper jaw bones during the earliest stages of palatal elongation. Growth of the maxillary anlage within the anterior secondary palate is uniquely coupled to RGZ-driven morphogenesis that more than doubles the length of this palatal segment prior to palatal shelf fusion. Our results also demonstrate that the future maxillary-palatine suture, approximated by the position ruga 1 and consistently associated with the palatine anlage, forms predominantly via the posterior differentiation of the maxilla within the expanding anterior secondary palate. Our complementary ontogenetic comparison of three inbred mouse strains identified small but significant strain-specific differences in early embryonic palatal segment contributions to the upper jaw. Although early palatal segment specific growth is not primarily responsible for adult differences in upper jaw morphology between these strains, our ontogenetic series of measurements provide a useful foundation for understanding the impact of background genetic effects on facial shape and elongation. In combination, our results provide a novel and particularly detailed picture of the earliest spatiotemporal dynamics of intramembranous midfacial skeletal specification and differentiation within the context of the surrounding palatal segment A-P elongation and associated rugae formation.

Unbiased transcriptome analysis of human cleft palate reveals evolutionally conserved molecular signatures of development: experimental study.

The development of the secondary palate, an essential process for hard palate formation, involves intricate cellular processes. Here, we examined the expression patterns of palatal fusion-associated genes in postdevelopmental human palatal tissues. Mucosal samples collected from the anterior fused (control; n=5) and posterior unfused regions (study; n=5) of cleft palate patients were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was conducted to identify consistent changes in molecular signaling pathways using hallmark (h) gene set collections from the Molecular Signature Database v7.4. The results of RNA sequencing were validated by epithelial-mesenchymal transition (EMT) assays with suppression of target genes, including lrp6, shh, Tgfβ-3 (Bioneer, Daejeon, Korea), and negative control siRNA in a human fibroblast cell line (hs68). Transcriptome profiling of the cleft mucosa demonstrated that the fully fused anterior mucosa exhibited globally upregulated EMT, Wnt β-catenin, Hedgehog, and TGF-β signaling pathways in gene set enrichment. This strongly indicates the evolutionary conserved similarities in pathways implicated in palatogenesis, as previously shown in murine models. In EMT assays with suppression of Lrp6, Shh, and TGF-β3 in human fibroblast cell lines, suppression of Lrp6 exhibited consistent suppression effects on EMT markers. This indicates a closer association with EMT compared to the other two signals. Our study highlights evolutionarily conserved molecular signatures and provides insights into the importance of the EMT pathway in palatal fusion in humans. Furthermore, intraindividual comparative analysis showed the spatial regulation of gene expression within the same organism. Further research and animal models are needed to explore the complexities of EMT-related palatal fusion.

Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6.

Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders. The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNβ gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNβ). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNβ promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M. NTD-IRF6 consists of a mix of α-helix and β-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNβ.

Exosomes Derived from Human Palatal Mesenchymal Cells Mediate Intercellular Communication During Palatal Fusion by Promoting Oral Epithelial Cell Migration.

Exosomes are important "messengers" in cell-cell interactions, but their potential effects on palatal fusion are still unknown. This study aimed to explore the role and mechanism of exosomes derived from palatal mesenchymal cells in epithelial-mesenchymal communication during palatogenesis. The expression of exosome marker CD63 and CD81 in palatal cells during palatogenesis was detected by immunofluorescence staining. After being purified from the supernatant of human embryonic palatal mesenchymal (HEPM) cells, exosomes (HEPM-EXO) were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot. HEPM-EXO were co-cultured with human immortalized oral epithelial cells (HIOEC). The effects of HEPM-EXO on the cell proliferation, migration, apoptosis and epithelial-mesenchymal transition (EMT) of HIOEC were evaluated. The proteins encapsulated in HEPM-EXO were analyzed by proteomic analysis. The extensive expression of CD63 and CD81 in palatal epithelial and mesenchymal cells were continuously detected during E12.5~E14.5, suggesting that exosomes were involved in the process of palatal fusion. The expression of CD63 was also observed in the acellular basement membrane between the palatal epithelium and the mesenchyme in vivo, and HEPM-EXO could be internalized by HIOEC in vitro, suggesting that exosomes are potent to diffuse through the cellular tissue boundary to mediate palatal cell-cell communication. Exposure of HEPM-EXO to HIOEC substantially inhibited the proliferation and stimulated the migration of HIOEC, but had no significant effect on cell apoptosis and EMT. Proteomic analysis revealed the basic characteristics of the proteins in HEPM-EXO and that exosomal THBS1 may potentially regulate the cell behaviors of HIOEC, which needs further verification. Gene ontology (GO) analysis uncovered that the proteins highly expressed in HEPM-EXO are closely related to wound healing, implying a promising therapeutic opportunity of HEPM-EXO in tissue injury treatment with future studies. HEPM-EXO mediated cell-cell communication by regulating cell proliferation and migration of oral epithelial cells during palatogenesis.

Research progress in molecular mechanism of palatal development

Cleft palate is one of the most common maxillofacial birth defects, which can occur alone or accompany with many known deformities. Palatal selves need to complete the process of vertical growth, elevation, adhesion and fusion in a specific time window of embryo development. Any abnormality in this process will lead to cleft palate. Although previous studies have identified many molecular networks that regulate the growth, location and fusion of palatal selves, there are still many unknown mechanisms for palatal development. The pathogenesis of cleft palate has not been clarified so far. In recent years, the molecular research on palate development has been deepened continuously. Here we summarize major recent advances and integrate the genes and molecular pathways with the cellular and morphogenetic processes of palatal shelf growth, patterning, elevation, adhesion, and fusion, in order to comprehensively understand the genotype-phenotype functional relationship and provide assistance in formulating effective prevention strategies for cleft palate disease.

Whole Exome Sequencing Identifies Damaging Variants in Indonesians with Clefts.

The interaction between genomics, genetic and environmental factors have been implicated in non-syndromic orofacial cleft development. In the current study, we investigated the contributions of rare and novel genetic variants in known cleft genes using whole exome sequencing (WES) data of Indonesians with non-syndromic orofacial clefts. WES was conducted on 6 individuals. Variants in their exons were called and annotated. These variants were filtered for novelty and rarity using MAF of 0 and 1%. Hospital in Indonesia. Indonesians with non-syndromic orofacial clefts. Deleterious variants were prioritized. Pathogenic amino acid changes effect on protein structure and function were identified using HOPE. Rare and novel variants in known cleft genes were filtered and their deleteriousness were predicted using polyphen, SIFT and CADD. We identified rare (MAF <1%) deleterious variants in 4 craniofacial genes namely MMACHC (rs371937044, MAF = 0.00011). SOS1 (rs190222208, MAF = 0.00045), TULP4 (rs199583035, MAF = 0.067), and MTHFD1L (rs143492706, MAF = 0.0044). MMACHC has a mouse knockout model with facial cleft and failure of palatal fusion. The individual with variant in MMACHC presented with nsCPO. Our study provides additional evidence for the role of TULP4, SOS1, MTHFD1L and MMACHC genes in nsOFC development. This is the first time MMACHC is implicated in nsOFC development in humans.

The roles of JAK2/STAT3 signaling in fusion of the secondary palate.

Cleft palate has a multifactorial etiology. In palatal fusion, the contacting medial edge epithelium (MEE) forms the epithelial seam, which is subsequently removed with the reduction of p63. Failure in this process results in a cleft palate. We herein report the involvement of janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in palatal fusion and that folic acid rescues the fusing defect by reactivating JAK2/STAT3. In closure of bilateral palatal shelves, STAT3 phosphorylation was activated at the fusing MEE and mesenchyme underlying the MEE. JAK2 inhibition by AG490 inhibited STAT3 phosphorylation and resulted in palatal fusion failure without removal of the epithelial seam, in which p63 and keratin 17 (K17) periderm markers were retained. Folic acid application restored STAT3 phosphorylation in AG490-treated palatal explants and rescued the fusion defect, in which the p63- and K17-positive epithelial seam were removed. The AG490-induced palatal defect was also rescued in p63 haploinsufficient explants. These findings suggest that JAK2/STAT3 signaling is involved in palatal fusion by suppressing p63 expression in MEE and that folate restores the fusion defect by reactivating JAK2/STAT3.

An improved multicellular human organoid model for the study of chemical effects on palatal fusion.

Tissue fusion is a mechanism involved in the development of the heart, iris, genital tubercle, neural tube, and palate during embryogenesis. Failed fusion of the palatal shelves could result in cleft palate (CP), a common birth defect. Organotypic models constructed of human cells offer an opportunity to investigate developmental processes in the human. Previously, our laboratory developed an organoid model of the human palate that contains human mesenchyme and epithelial progenitor cells to study the effects of chemicals on fusion. Here, we developed an organoid model more representative of the embryonic palate that includes three cell types: mesenchyme, endothelial, and epithelial cells. We measured fusion by a decrease in epithelial cells at the contact point between the organoids and compared the effects of CP teratogens on fusion and toxicity in the previous and current organoid models. We further tested additional suspect teratogens in our new model. We found that the three-cell-type model is more sensitive to fusion inhibition by valproic acid and inhibitors of FGF, BMP, and TGFβRI/II. In this new model, we tested other suspect CP teratogens and found that nocodazole, topiramate, and Y27632 inhibit fusion at concentrations that do not induce toxicity. This sensitive human three-cell-type organotypic model accurately evaluates chemicals for cleft palate teratogenicity.

Gene Regulatory Networks and Signaling Pathways in Palatogenesis and Cleft Palate: A Comprehensive Review.

Palatogenesis is a complex and intricate process involving the formation of the palate through various morphogenetic events highly dependent on the surrounding context. These events comprise outgrowth of palatal shelves from embryonic maxillary prominences, their elevation from a vertical to a horizontal position above the tongue, and their subsequent adhesion and fusion at the midline to separate oral and nasal cavities. Disruptions in any of these processes can result in cleft palate, a common congenital abnormality that significantly affects patient's quality of life, despite surgical intervention. Although many genes involved in palatogenesis have been identified through studies on genetically modified mice and human genetics, the precise roles of these genes and their products in signaling networks that regulate palatogenesis remain elusive. Recent investigations have revealed that palatal shelf growth, patterning, adhesion, and fusion are intricately regulated by numerous transcription factors and signaling pathways, including Sonic hedgehog (Shh), bone morphogenetic protein (Bmp), fibroblast growth factor (Fgf), transforming growth factor beta (Tgf-β), Wnt signaling, and others. These studies have also identified a significant number of genes that are essential for palate development. Integrated information from these studies offers novel insights into gene regulatory networks and dynamic cellular processes underlying palatal shelf elevation, contact, and fusion, deepening our understanding of palatogenesis, and facilitating the development of more efficacious treatments for cleft palate.

High throughput miRNA sequencing and bioinformatics analysis identify the mesenchymal cell proliferation and apoptosis related miRNAs during fetal mice palate development.

Palatogenesis requires a precise spatiotemporal regulation of gene expression. Recent studies indicate that microRNAs (miRNAs) are key factors in normal palatogenesis. The present study aimed to explain the regulatory mechanisms of miRNAs during palate development. Pregnant ICR mice were choose at embryonic day 10.5 (E10.5). Hemotoxylin and eosin (H&E) staining was used to observe the morphological changes during the development of palatal process at embryonic day (E)13.5, E14.0, E14.5, E15.0 and E15.5. The fetal palatal tissues were collected at E13.5, E14.0, E14.5 and E15.0 to explore miRNA expression and function by high throughput sequencing and bioinformatic analysis. Mfuzz cluster analysis was used to look for miRNAs related to the fetal mice palate formation. The target genes of miRNAs were predicted by miRWalk. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed base on target genes. The mesenchymal cell proliferation and apoptosis related miRNAs-genes networks were predicted and constructed using miRWalk and Cytoscape software. The expression of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was detected by a quantitative real-time PCR (RT-qPCR) assay. H&E staining found that the palatal process grows vertically along the sides of the tongue at E13.5, the position of the tongue begins to descend and the bilateral palatal processes rise above the tongue at E14.0, the palatal process grows horizontally at E14.5, there is palatal contact fusion at E15.0, and the palatal suture disappeared at E15.5. Nine clusters of miRNA expression changes were identified in the fetal mice palate formation progression, including two reducing trends, two rising trends and five disordered trends. Next, the heatmap showed the miRNA expression from Clusters 4, 6, 9, 12 in the E13.5, E14.0, E14.5 and E15.0 groups. GO functional and KEGG pathway enrichment analysis found target genes of miRNAs in clusters involved in regulation of mesenchymal phenotype and the mitogen-activated protein kinase (MAPK) signaling pathway. Next, mesenchymal phenotype related miRNA-genes networks were constructed. The heatmap showing that the mesenchymal phenotype related miRNA expression of Clusters 4, 6, 9 and 12 at E13.5, E14.0, E14.5 and E15.0. Furthermore, the mesenchymal cell proliferation and apoptosis related miRNA-gene networks were identified in Clusters 6 and 12, including mmu-miR-504-3p-Hnf1b, etc. The expression level of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was verified by a RT-qPCR assay. For the first time, we identified that clear dynamic miRNA expression during palate development. Furthermore, we demonstrated that mesenchymal cell proliferation and apoptosis related miRNAs, genes and the MAPK signaling pathway are important during fetal mice palate development.

Canonical Wnt signaling is not required for Tgfb3 expression in the basal medial edge epithelium during palatogenesis.

The secondary palate forms from two lateral primordia called the palatal shelves which form a contact in the midline, become adherent at the fusing interface (medial edge epithelia, MEE) and subsequently fuse. The gene encoding transforming growth factor-ß3 (Tgfb3) is strongly and specifically expressed in MEE cells. Our previous study suggested that Tgfb3 expression is controlled via upstream cis-regulatory elements in and around the neighboring Ift43 gene. Another study suggested that the canonical Wnt signaling via ß-Catenin is responsible for the MEE-specific Tgfb3 gene expression, since deletion of the Ctnnb1 gene by a commonly used Keratin 14-Cre (K14Cre) mouse line almost completely abolished Tgfb3 expression in the MEE resulting in cleft palate. Here, we wanted to analyze whether Tcf/Lef consensus binding sites located in the previously identified regions of the Ift43 gene are responsible for the spatiotemporal control of Tgfb3 expression during palatogenesis. We show that contrary to the previous report, deletion of the Ctnnb1 gene in basal MEE cells by the K14Cre driver (the same K14Cre mouse line was used as in the previous study referenced above) does not affect the MEE-specific Tgfb3 expression or TGFß3-dependent palatal epithelial fusion. All mutant embryos showed a lack of palatal rugae accompanied by other craniofacial defects, e.g., a narrow snout and a small upper lip, while only a small subset (<5%) of Ctnnb1 mutants displayed a cleft palate. Moreover, the K14Cre:Ctnnb1 embryos showed reduced levels and altered patterns of Shh expression. Our present data imply that epithelial ß-catenin may not be required for MEE-specific Tgfb3 expression or palatal epithelial fusion.

Live imaging observation of elevation of the anterior palatal shelf in mouse embryos.

The mammalian secondary palate develops through complex processes including palatal shelf growth, elevation, and fusion. Palatal shelf elevation is a process accompanied by large-scale morphological changes over a short period. The elevation pattern changes along the anterior-posterior axis; the anterior region elevates by the "flip-up" model, and the middle and posterior regions reorient through the "flow" model; however, the mechanisms of both models are unclear because of the rapid progression of the elevation in utero. To observe palatal elevation in real-time in detail, we aimed to establish a live imaging method using explants of the anterior region of the palatal shelf in mouse embryos before the beginning of elevation. Changes in the degree of shelf orientation were measured, which showed that the palatal shelf continuously changed shape toward the lingual side. The changes in the angle between the lingual and buccal bases of the palatal shelf were different; the morphological change in the lingual side was more acute, and the buccal side was more obtuse. The timing of morphological changes of the lingual and buccal sides was nearly simultaneous, suggesting that the anterior region of the palatal shelf in vitro elevated according to the "flip-up" model. This live imaging method enables the continuous observation of palatal shelf elevation and provides new insights into palatogenesis. This article is protected by copyright. All rights reserved.

EARLY INTERVENTION OF SPEECH AND LANGUAGE DEVELOPMENT PROBLEMS IN CHILDREN WITH CLEFT LIP AND PALATE

Children with cleft lip and palate (CLP) can have language and speech difficulties. This happens because of changes in the orofacial anatomical structure, so there are some limitations in articulation abilities. In addition to speech-sound disorders, children with cleft lip and palate often experience problems with language skills due to a limited vocabulary as well as syntactic and morphological abilities. Children with CLP can experience problems with sentence structure and speech speed (words per minute), so children with cleft lip and palate often experience language delays. The research conducted is a systematic research review. This research was conducted by searching for and selecting five types of data from the results of previous studies and from the results of clinical trials conducted on different ethnicities, races, and locations around the world. Research information was retrieved from databases, including PubMed, Wiley, and Google Scholar. The data analysis technique used in this systematic review is the data extraction method. The results showed that the search for articles that matched the searches and explanations about early handling of language development in children with cleft lip and palate, namely. Early intervention with parental assistance. There were five articles on the results of interventions in children with cleft lip and palate, namely, early intervention after the cleft lip and palate fusion process and educational support for parents.

Increased miR-200c levels disrupt palatal fusion by affecting apoptosis, cell proliferation, and cell migration

The mammalian palate separates the oral and nasal cavities, facilitating proper feeding, respiration, and speech. Palatal shelves, composed of neural crest-derived mesenchyme and surrounding epithelium, are a pair of maxillary prominences contributing to this structure. Palatogenesis reaches completion upon the fusion of the midline epithelial seam (MES) following contact between medial edge epithelium (MEE) cells in the palatal shelves. This process entails numerous cellular and molecular occurrences, including apoptosis, cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT). MicroRNAs (miRs) are small, endogenous, non-coding RNAs derived from double-stranded hairpin precursors that regulate gene expression by binding to target mRNA sequences. Although miR-200c is a positive regulator of E-cadherin, its role in palatogenesis remains unclear. This study aims to explore the role of miR-200c in palate development. Before contact with palatal shelves, mir-200c was expressed in the MEE along with E-cadherin. After palatal shelf contact, miR-200c was present in the palatal epithelial lining and epithelial islands surrounding the fusion region but absent in the mesenchyme. The function of miR-200c was investigated by utilizing a lentiviral vector to facilitate overexpression. Ectopic expression of miR-200c resulted in E-cadherin upregulation, impaired dissolution of the MES, and reduced cell migration for palatal fusion. The findings imply that miR-200c is essential in palatal fusion as it governs E-cadherin expression, cell death, and cell migration, acting as a non-coding RNA. This study may contribute to clarifying the underlying molecular mechanisms in palate formation and provides insights into potential gene therapies for cleft palate.

DNA methylation differences in monozygotic twins with Van der Woude syndrome.

Van der Woude Syndrome (VWS) is an autosomal dominant disorder responsible for 2% of all syndromic orofacial clefts (OFCs) with IRF6 being the primary causal gene (70%). Cases may present with lip pits and either cleft lip, cleft lip with cleft palate, or cleft palate, with marked phenotypic discordance even among individuals carrying the same mutation. This suggests that genetic or epigenetic modifiers may play additional roles in the syndrome's etiology and variability in expression. We report the first DNA methylation profiling of 2 pairs of monozygotic twins with VWS. Our goal is to explore epigenetic contributions to VWS etiology and variable phenotypic expressivity by comparing DNAm profiles in both twin pairs. While the mutations that cause VWS in these twins are known, the additional mechanism behind their phenotypic risk and variability in expression remains unclear. We generated whole genome DNAm data for both twin pairs. Differentially methylated positions (DMPs) were selected based on: (1) a coefficient of variation in DNAm levels in unaffected individuals < 20%, and (2) intra-twin pair absolute difference in DNAm levels >5% (delta beta > | 0.05|). We then divided the DMPs in two subgroups for each twin pair for further analysis: (1) higher methylation levels in twin A (Twin A > Twin B); and (2) higher methylation levels in twin B (Twin B >Twin A). Gene ontology analysis revealed a list of enriched genes that showed significant differential DNAm, including clef-associated genes. Among the cleft-associated genes, TP63 was the most significant hit (p=7.82E-12). Both twin pairs presented differential DNAm levels in CpG sites in/near TP63 (Twin 1A > Twin 1B and Twin 2A < Twin 2B). The genes TP63 and IRF6 function in a biological regulatory loop to coordinate epithelial proliferation and differentiation in a process that is critical for palatal fusion. The effects of the causal mutations in IRF6 can be further impacted by epigenetic dysregulation of IRF6 itself, or genes in its pathway. Our data shows evidence that changes in DNAm is a plausible mechanism that can lead to markedly distinct phenotypes, even among individuals carrying the same mutation.

Periderm Fate during Palatogenesis: TGF-β and Periderm Dedifferentiation

Failure of palatogenesis results in cleft palate, one of the most common congenital disabilities in humans. During the final phases of palatogenesis, the protective function of the peridermal cell layer must be eliminated for the medial edge epithelia to adhere properly, which is a prerequisite for the successful fusion of the secondary palate. However, a deeper understanding of the role and fate of the periderm in palatal adherence and fusion has been hampered due to a lack of appropriate periderm-specific genetic tools to examine this cell type in vivo. Here we used the cytokeratin-6A (Krt-6a) locus to develop both constitutive (Krt6ai-Cre) and inducible (Krt6ai-Cre ERT2 ) periderm-specific Cre driver mouse lines. These novel lines allowed us to achieve both the spatial and temporal control needed to dissect the periderm fate on a cellular resolution during palatogenesis. Our studies suggest that, already before the opposing palatal shelves contact each other, at least some palatal periderm cells start to gradually lose their squamous periderm-like phenotype and dedifferentiate into cuboidal cells, reminiscent of the basal epithelial cells seen in the palatal midline seam. Moreover, we show that transforming growth factor–β (TGF-β) signaling plays a critical periderm-specific role in palatogenesis. Thirty-three percent of embryos lacking a gene encoding the TGF-β type I receptor (Tgfbr1) in the periderm display a complete cleft of the secondary palate. Our subsequent experiments demonstrated that Tgfbr1-deficient periderm fails to undergo appropriate dedifferentiation. These studies define the periderm cell fate during palatogenesis and reveal a novel, critical role for TGF-β signaling in periderm dedifferentiation, which is a prerequisite for appropriate palatal epithelial adhesion and fusion.

Making the head: Caspases in life and death.

The term apoptosis, as a way of programmed cell death, was coined a half century ago and since its discovery the process has been extensively investigated. The anatomy and physiology of the head are complex and thus apoptosis has mostly been followed in separate structures, tissues or cell types. This review aims to provide a comprehensive overview of recent knowledge concerning apoptosis-related molecules involved in the development of structures of head with a particular focus on caspases, cysteine proteases having a key position in apoptotic pathways. Since many classical apoptosis-related molecules, including caspases, are emerging in several non-apoptotic processes, these were also considered. The largest organ of the head region is the brain and its development has been extensively investigated, including the roles of apoptosis and related molecules. Neurogenesis research also includes sensory organs such as the eye and ear, efferent nervous system and associated muscles and glands. Caspases have been also associated with normal function of the skin and hair follicles. Regarding mineralised tissues within craniofacial morphogenesis, apoptosis in bones has been of interest along with palate fusion and tooth development. Finally, the role of apoptosis and caspases in angiogenesis, necessary for any tissue/organ development and maintenance/homeostasis, are discussed. Additionally, this review points to abnormalities of development resulting from improper expression/activation of apoptosis-related molecules.

Using whole exome sequencing to identify susceptibility genes associated with nonsyndromic cleft lip with or without cleft palate.

Cleft lip and palate is a common congenital birth defect in humans. Its incidence rate in China is as high as 1.82%, and is now a frequent deformity observed among the Chinese population; moreover, it varies across regions. Although the etiology of nonsyndromic cleft lip with or without cleft palate (NSCL/P) has been widely investigated, the results are inconsistent. The specific genes and mechanisms responsible for NSCL/P have not been fully understood. Whole exome sequencing (WES) is a new strategy for studying pathogenic genes. WES studies on NSCL/P have not been conducted in East China. Therefore, the aim of this study was to screen candidate genes of NSCL/P in East China using WES and analyze the temporal and spatial expressions of the candidate genes during embryonic palatal development. WES was performed in 30 children with NSCL/P from East China to screen candidate genes. A bioinformatics analysis was performed using commercially available software. Variants detected by WES were validated by immunohistochemistry and western blotting. After WES, 506,144 single-nucleotide variant sites were found. The results of database comparison, functional analysis, and mass spectrometry revealed that only the laminin alpha 5 (LAMA5) gene (site: rs145192286) was associated with NSCL/P. Immunohistochemistry results showed that LAMA5 expression in the medial edge epithelium changed with formation, lifting, and contact during palatogenesis. Almost no LAMA5 expression was detected in the palatal mesenchyme or after palatal fusion. Western blotting and immunohistochemistry results showed consistent trends. In conclusion, the WES results shows that the mutation at the site (rs145192286) of LAMA5 is associated with NSCL/P. The temporal and spatial expressions of LAMA5 during palatal development further demonstrate the involvement of this gene. Therefore, we speculate that LAMA5 is a new candidate pathogenic gene of NSCL/P. The identification of new pathogenic genes would help elucidate the pathogenesis of NSCL/P and provide a scientific basis for the prenatal diagnosis, prevention, and treatment of NSCL/P.