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.

Many processes take place during embryogenesis, and the development of the palate mainly involves proliferation, migration, osteogenesis, and epithelial-mesenchymal transition. Abnormalities in any of these processes can be the cause of cleft palate (CP). There have been few reports on whether C-X-C motif chemokine receptor 4 (CXCR4), which is involved in embryonic development, participates in these processes. In our study, the knockdown of Cxcr4 inhibited the migration of mouse embryonic palatal mesenchymal (MEPM) cells similarly to the use of its inhibitor plerixafor, and the inhibition of cell migration in the Cxcr4 knockdown group was partially reversed by supplementation with C-X-C motif chemokine ligand 12 (CXCL12). In combination with low-dose retinoic acid (RA), plerixafor increased the incidence of cleft palates in mice by decreasing the expression of Cxcr4 and its downstream migration-regulating gene Rac family small GTPase 1 (RAC1) mediating actin cytoskeleton to affect lamellipodia formation and focal complex assembly and ras homolog family member A (RHOA) regulating the actin cytoskeleton to affect stress fiber formation and focal complex maturation into focal adhesions. Our results indicate that the disruption of cell migration and impaired normal palatal development by inhibition of Cxcr4 expression might be mediated through Rac1 with RhoA. The combination of retinoic acid and plerixafor might increase the incidence of cleft palate, which also provided a rationale to guide the use of the drug during conception.

Cleft lip and palate is one of the most frequent congenital developmental defects. Autophagy is a highly conserved process of cell self-degradation in eukaryotes, involving multiple biological processes in which chloroquine (CQ) is the most common inhibitor. However, whether CQ affects and how it affects palate development is unknown. Mouse embryonic palatal cells (MEPCs) were treated with CQ to observe cell viability, apoptosis, migration, osteogenic differentiation by cell proliferation assay, flow cytometric analysis, scratch assay, and alizarin red staining. PI staining was used to measure cell cycle distribution. Immunofluorescence (IF) assay and transmission electron microscopy were used to detect autophagosomes. The autophagy-related factors (LC3 and P62), apoptosis-related markers (P53, caspase-3 cleaved caspase-3, BAX, and BCL-2), and cell cycle-related proteins (P21, CDK2, CDK4, cyclin D1, and cyclin E) were all measured by western blot. CQ inhibited the proliferation of MEPCs by arresting the G0/G1 phase of the cell cycle in a concentration- and time-dependent manner with cell cycle-related proteins P21 upregulated and CDK2, CDK4, cyclin D1, and cyclin E downregulated. Then we detected CQ also induced cell apoptosis in a dose-dependent manner by decreasing the BCL-2/BAX ratio and increasing cleaved caspase-3. Next, it was investigated that migration and osteogenesis of MEPCs decreased with CQ treatment in a dose-dependent manner. Meanwhile, CQ blocked the autophagy pathway by upregulating LC3II and P62 expressions which activated the P53 pathway. CQ activates P53 which affects MEPC biological characteristics by changing the proliferation and apoptosis of MEPCs through inhibiting autophagy.

Cleft palate is one of the most frequent craniofacial malformation birth defects. Miniature pigs (Sus scrofa) are a valuable alternative large animal model to explore human palate development. Presently, the microRNA (miRNA) expression profiles in miniature pigs during palatogenesis from embryonic day (E) 30 to 50 were identified. A total of 2044 known miRNAs and 192 novel miRNAs were identified. The functional characteristics of their potential target genes were identified using Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway analysis. MiRNAs displayed diverse expression levels among the different stages. Using Short Time-series Expression Miner software to investigate the expression patterns of miRNAs from E30-50, all miRNAs were clustered into 20 profiles. The profiles showing miRNAs expression decreased (profile 0)/increased (profile 19) from E30-50 were the main patterns during palatogenesis. Hub genes of four significant modules were identified by weighted correlation network analysis, including ssc-miR-98, ssc-miR-27a_R + 1, and ssc-miR-150, etc. which might be novel potential targets for regulating palate development. The data are expected to improve the understanding of palate development and the etiology of cleft palate in further studies.

Normal mammalian palatogenesis is a complex process that requires the occurrence of a tightly regulated series of specific and sequentially regulated cellular events. Cleft lip/palate (CLP), the most frequent craniofacial malformation birth defects, may occur if any of these events undergo abnormal interference. Such defects not only affect the patients, but also pose a financial risk for the families. In our recent study, the miniature pig was shown to be a valuable alternative large animal model for exploring human palate development by histology. However, few reports exist in the literature to document gene expression and function during swine palatogenesis. To better understand the genetic regulation of palate development, an mRNA expression profiling analysis was performed on miniature pigs, Sus scrofa. Five key developmental stages of miniature pigs from embryonic days (E) 30-50 were selected for transcriptome sequencing. Gene expression profiles in different palate development stages of miniature pigs were identified. Nine hundred twenty significant differentially expressed genes were identified, and the functional characteristics of these genes were determined by gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Some of these genes were associated with HH (hedgehog), WNT (wingless-type mouse mammary tumor virus integration site family), and MAPK (mitogen-activated protein kinase) signaling, etc., which were shown in the literature to affect palate development, while some genes, such as HIP (hedgehog interacting protein), WNT16, MAPK10, and LAMC2 (laminin subunit gamma 2), were additions to the current understanding of palate development. The present study provided a comprehensive analysis for understanding the dynamic gene regulation during palate development and provided potential ideas and resources to further study normal palate development and the etiology of cleft palate.

During early palate development, gene expression and regulation exhibit heterogeneity along the anterior-posterior axis. Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways play essential roles in secondary palatal formation but the exact relationship between the TGF-β and BMP pathways in palate development remains unknown. Here, we demonstrate that, during early secondary palate development, phospho-(p)Smad1/5/8 is highly expressed in the anterior palate but relatively lowly expressed in the posterior palate. Conversely, pSmad2/3 has a lower expression level in the anterior palate than in the posterior palate. With the BRE-Gal reporter, we found that the canonical BMP signaling pathway was not activated in the anterior palate but exhibited a moderate level in the posterior palate. Co-immunoprecipitation revealed that Smad4 bound to pSmad1/5/8 only in the posterior palate and not in the anterior palate. Knocking-out of Tgfbr2 (Wnt1-Cre;Tgfbr2 f/f;BRE) in the palatal mesenchyme enhanced canonical BMP activity in the posterior palate but not in the anterior palate, because of decreased pSmad2/3. pSmad1/5/8-Smad4 complexes were found to be dramatically increased in posterior palatal mesenchymal cells at embryonic day 13.5 cultured in the presence of SB525334. Proximity ligation assay also showed pSmad1/5/8-Smad4 complexes were increased in the posterior palate of Wnt1-Cre;Tgfbr2 f/f mice. Therefore, the reduction of pSmad2/3 level in the palatal mesenchyme of Wnt1-Cre;Tgfbr2 f/f;BRE mice contributes primarily to the increase of pSmad1/5/8-Smad4 complexes leading to enhanced canonical BMP activity in the posterior palate. Moreover, during early development, canonical BMP signaling operates in the posterior palate but is completely absent in the anterior palate. Canonical TGF-β signaling suppresses canonical BMP signaling activity in the posterior palate by competing limited Smad4.

Palate development is an important morphogenetic event in facial development, including the fusion of the lateral and medial nasal portions of the frontonasal process and maxilla. Derailments of any of these events may result in cleft palate, the most frequent congenital craniofacial abnormality. Recent research has shown that the microanatomy of the miniature pig oral maxillofacial region is quite similar to that of humans, and the use of miniature pigs as a large animal model for dental and orofacial research is increasing. Little information is available, however, about the development of the miniature pig palate. Here, using histological and ultrastructural methods, we describe the developmental stages of the palate in miniature pigs. Sections from E26, E30, E35, E40, E45, and E50 embryos were stained with hematoxylin-eosin, and selected specimens were also processed for electron microscopy. The development of the miniature pig palate can be divided into four stages: growth of the bilateral palatal shelves alongside the tongue at E30; elevation of the horizontal position above the tongue at E35; establishment of bilateral shelf contact at the midline from E35-50; and a final fusion step at E50, similar to the mouse and human. The histological characteristics of the miniature pig palate at different developmental stages were synchronously verified at the ultrastructural level. Our study provides a piece of first-hand data regarding palate morphological organogenesis in the miniature pig and a foundation for further research with this model to explore mechanisms of cleft palate development. Anat Rec, 300:1409-1419, 2017. © 2017 Wiley Periodicals, Inc.