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Bone Morphogenetic Protein (BMP) signaling is essential for craniofacial development, though little is known about the mechanisms that govern BMP secretion. We show that depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchyme. We show endogenous transient changes in intracellular calcium occur in cranial neural crest cells, the cells from which embryonic palate mesenchyme derives. Waves of transient changes in intracellular calcium suggest that these cells are electrically coupled and may temporally coordinate BMP release. These transient changes in intracellular calcium persist in palate mesenchyme cells from embryonic day 9.5 to 13.5 mice. Disruption of a potassium channel called Kcnj2 significantly decreases the amplitude of calcium transients and the ability of cells to secrete BMP. Kcnj2 knockout mice have cleft palate and reduced BMP signaling. Our data suggest that temporal control of developmental cues is regulated by ion channels, depolarization, and intracellular calcium for mammalian craniofacial morphogenesis. BMP signaling is important for multiple process, including craniofacial development, but relatively little is known about how BMP ligands are secreted. Here they show that embryonic palate mesenchyme cells undergo transient changes in intracellular calcium and that depolarization of these cells induces BMP4 release, suggesting that ion channels are a node in BMP4 signaling.

Little is known about the regulation and function of phase separation in craniofacial developmental disorders. MSX1 mutations are associated with human cleft palate, the most common craniofacial birth defect. Here, we show that MSX1 phase separation is a vertebrate-conserved mechanism underlying embryonic palatal fusion. Notably, MSX1 phase separation is triggered by its intrinsically disordered protein region (IDR) and regulated by PRMT1-catalyzed methylation, specifically asymmetric dimethylation of arginine in the MSX1 IDR including R150 and R157. Hypomethylated MSX1 due to methylation site mutations and PRMT1 deficiency consistently leads to abnormal MSX1 phase separation to form less dynamic gel-like condensates, resulting in proliferation defects of embryonic palatal mesenchymal cells and cleft palate. Besides, high frequency mutations in the MSX1 IDR, especially R157S, have been identified in humans with cleft palate. Overall, we reveal the function and regulatory pathway of MSX1 phase separation as a conserved mechanism underlying cleft palate, providing a proof-of-concept example of a phenotype-associated phase separation mechanism associated with craniofacial developmental disorders. MSX1 phase separation are associated with cleft palate. Here the authors show that MSX1 phase separation, triggered by its N-terminal intrinsically disordered region and regulated by PRMT1-catalyzed methylation, is a vertebrate-conserved mechanism for embryonic palatal development.

Background Overconsumption of retinoic acid (RA) or its analogues/derivatives has been linked to severe craniomaxillofacial malformations, such as cleft palate and midface hypoplasia. It has been noted that RA disturbed the proliferation and migration of embryonic palatal mesenchymal (EPM) cells in these malformations, yet the exact mechanisms underlying these disruptions remained unclear. Methods A model of retinoic acid (RA)-induced cleft palate in fetal mice was successfully established. Histological alterations in the palate were evaluated using Hematoxylin and Eosin (H&E) staining and RNA in situ hybridization (RNAscope). Cellular proliferation levels were quantified via the Cell Counting Kit-8 (CCK-8) assay and EdU incorporation assay, while cell migration capabilities were investigated using wound healing and Transwell assays. Mitochondrial functions were assessed through Mito-Tracker fluorescence, mitochondrial reactive oxygen species (ROS) measurement, ATP level quantification, and mitochondrial DNA (mtDNA) copy number analysis. Differential gene expression and associated signaling pathways were identified through bioinformatics analysis. Alterations in the transcriptional and translational levels of Lhx6 and genes associated with mitophagy were quantified using quantitative PCR (qPCR) and Western blot analysis, respectively. Mitochondrial morphology and the mitochondrial autophagosomes within cells were examined through transmission electron microscopy (TEM). Results Abnormal palatal development in mice, along with impaired proliferation and migration of human embryonic palatal mesenchymal (HEPM) cells, was associated with RA affecting mitochondrial function and concomitant downregulation of Lhx6. Knockdown of Lhx6 in HEPM cells resulted in altered cell proliferation, migration, and mitochondrial function. Conversely, the aberrant mitochondrial function, proliferation, and migration observed in RA-induced HEPM cells were ameliorated by overexpression of Lhx6. Subsequent research demonstrated that Lhx6 ameliorated RA-induced dysfunction in HEPM cells by modulating PINK1/Parkin-mediated mitophagy, thereby activating the MAPK signaling pathways. Conclusion Lhx6 is essential for mitochondrial homeostasis via tuning PINK1/Parkin-mediated mitophagy and MAPK signaling pathways. Downregulation of Lhx6 by RA transcriptionally disturbs the mitochondrial homeostasis, which in turn leads to the proliferation and migration defect in HEPM cells, ultimately causing the cleft palate. Graphical abstract

Cleft palate only (CPO) is a multifactorial craniofacial malformation with significant genetic and epigenetic contributions. Among these, microRNAs (miRNAs) have emerged as key regulators of palate development, although their alterations in CPO remain incompletely characterized. In this study, we performed a comprehensive miRNA expression analysis on palatal tissues from an Italian cohort of non-syndromic CPO patients, compared with a human embryonic palatal mesenchymal (HEPM) cell line. Using the NanoString® nCounter® platform for miRNA profiling, we identified significant deregulation of several miRNAs, notably the upregulation of miR-205-5p and miR-200c-3p and the downregulation of miR-125a-5p in CPO tissues. Based on these expression changes, a functional analysis was carried out to identify potential target genes. Validation in primary cell cultures derived from patient tissues confirmed these expression patterns. Functional analyses and target predictions implicated PAX9, a key transcription factor essential for palatogenesis, as a probable target of miR-205-5p, while miR-125a-5p was associated with the regulation of PRTG and PRSS35—genes involved in neural crest cell biology and extracellular matrix remodeling, respectively. Although modulation of certain predicted targets of miR-200c-3p was observed, in vitro inhibition experiments did not show significant changes in gene expression, highlighting the complexity of miRNA regulatory networks and the need for further studies to unravel these interactions. These findings identify miRNA alterations associated with CPO tissue and fibroblasts, highlighting novel candidate pathways for further mechanistic and therapeutic investigation.

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.

Cleft palate is one of the most common congenital abnormalities and one of the main symptoms of Stickler syndrome. Secondary palate development is a complex multi-step process that involves raising the palatal frame from a vertical to a horizontal position. Lysyl oxidase-like 3 (LOXL3), a member of the lysyl oxidase family responsible for the crosslinking in collagen, is also one of the mutated genes detected in Stickler syndrome. Loss of Loxl3 causes delayed palatal shelf elevation, which in turn resulted in cleft palate. However, the precise mechanisms of palatal shelf delayed elevation remain unclear. In this study, we deeply investigated the mechanism of Loxl3 induced delayed elevation in palatal shelves. We found that Loxl3 deficiency caused reduced cell proliferation in both medial and posterior palatal mesenchyme through BrdU labeling and Western blot analysis (p < 0.05, p < 0.01), decreased migration of palatal mesenchymal cells through cell scratch assay (p < 0.05), and decreased expression of genes associated with proliferation through Western blot analysis (p < 0.05, p < 0.01) at E14. We found that the specific deletion of Loxl3 in the palatal mesenchyme resulted in delayed elevation but normal fusion of palatal shelves, also reduced cell proliferation and collagen fibers deposition in medial palatal mesenchyme through BrdU labeling and histological analysis (p < 0.05, p < 0.01). Thus, our data suggest that Loxl3 regulates cell proliferation and collagen fibers deposition in the palatal mesenchyme, thus controlling palatal shelf elevation.

Despite advances in understanding the morphological disruptions that lead to defects in palate formation, the precise perturbations within the signaling microenvironment of palatal clefts remain poorly understood. To explore in greater depth the genomic basis of palatal clefts, we designed and implemented the first single cell spatial RNA-sequencing study in a cleft palate model, utilizing the Pax9 −/− murine model at multiple developmental timepoints, which exhibits a consistent cleft palate defect. Visium HD, an emerging platform for true single-cell resolution spatially resolved transcriptomics, was employed using custom bins of 2 × 2 μm spatial gene expression data. Validation of spatial gene expression was then validated using custom designed Xenium In Situ mRNA spatial profiling and RNAscope Multiplex assays. Functional enrichment analysis revealed a palate cell-specific perturbation in Wnt signaling effector function in tandem with disrupted expression of extracellular matrix genes in developing mesenchyme. As a key step toward laying the framework for identifying key molecular targets these data can be used for translational studies aimed at developing effective therapies for human palatal clefts.

Rupture of the basement membrane in fused palate tissue can cause the palate to separate after fusion in mice, leading to the development of cleft palate. Here, we further elucidate the mechanism of palatal separation after palatal fusion in 8–10-week-old ICR female mice. On day 12 of gestation, 40 μg/kg of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), sufficient to cause cleft palate in 100% of mice, was dissolved in 0.4 mL of olive oil containing toluene and administered as a single dose via a gastric tube. Fetal palatine frontal sections were observed by H&E staining, and epithelial cell adhesion factors, apoptosis, and cell proliferation were observed from the anterior to posterior palate. TUNEL-positive cells and Ki67-positive cells were observed around the posterior palatal dissection area of the TCDD-treated group. Moreover, in fetal mice exposed to TCDD, some fetuses exhibited cleft palate dehiscence during fusion. The results suggest that palatal dehiscence may be caused by abnormal cell proliferation in epithelial tissues, decreased intercellular adhesion, and inhibition of mesenchymal cell proliferation. By elucidating the mechanism of cleavage after palatal fusion, this research can contribute to establishing methods for the prevention of cleft palate development.

Abnormal embryonic development leads to the formation of cleft palate (CP) which is difficult to be detected by genetic screening and needs sequent treatment from infants to adults. There are no interceptive treatment about CP until now. Germline deletion of phosphatase and tensin homolog ( Pten ) was related to embryonic malformation and regulated tumor cell proliferation through glycolysis. However, the role of Pten in CP and the relationship between CP, Pten, and glycolysis are unknown. In our research, we constructed Pten knockdown models in vitro and in vivo. Our results provided preliminary evidence that blocking Pten by its inhibitor such as VO-OHpic might be an effective interceptive treatment in early period of palate development when pregnant mother expose in harmful environment during the early period of palate development to reducing CP occurring which was related with the crosstalk between Pten , and glycolysis in the process. Graphical Abstract

Cleft palate (CP) is a common congenital birth defect. Cellular and morphological processes change dynamically during palatogenesis, and any disturbance in this process could result in CP. However, the molecular mechanisms steering this fundamental phase remain unclear. One study suggesting a role for miRNAs in palate development via maternal small extracellular vesicles (SEVs) drew our attention to their potential involvement in palatogenesis. In this study, we used an in vitro model to determine how SEVs derived from amniotic fluid (ASVs) and maternal plasma (MSVs) influence the biological behaviors of mouse embryonic palatal mesenchyme (MEPM) cells and medial edge epithelial (MEE) cells; we also compared time-dependent differential expression (DE) miRNAs in ASVs and MSVs with the DE mRNAs in palate tissue from E13.5 to E15.5 to study the dynamic co-regulation of miRNAs and mRNAs during palatogenesis in vivo. Our results demonstrate that some pivotal biological activities, such as MEPM proliferation, migration, osteogenesis, and MEE apoptosis, might be directed, in part, by stage-specific MSVs and ASVs. We further identified interconnected networks and key miRNAs such as miR-744-5p, miR-323-5p, and miR-3102-5p, offering a roadmap for mechanistic investigations and the identification of early CP biomarkers.