Search

SUMMARYThe abnormal perturbation in gene regulation during palatogenesis may lead to cleft palate, a major congenital birth defect in humans and mice. However, a comprehensive multi-omic map of the developing secondary palate at single-cell resolution is lacking. In this study, we performed single-cell multiome sequencing and profiled chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. Application of optimal transport reconstructed five trajectories, representing continuous differentiation of multipotent cells into different subpopulations in later stages. By linking open chromatin signals to gene expression changes, we discovered a list of lineage-determining transcription factors, such as Shox2 for the anterior and Dlx1/2 for the posterior palatal mesenchymal trajectories. In conclusion, this study charted epigenetic and transcriptional dynamics during palatogenesis, which provides a valuable resource for the community and facilitate future research in cleft palate.HighlightsThe first report on building a single-cell multi-omics atlas with joint chromatin accessibility and gene expression measurements from the same cells during the development of mouse secondary palate.Application of optimal transport calculated fate probabilities to different terminal states and recovered continuous landscapes during mouse secondary palate development.By linkingcis-regulatory DNA elements to target genes, we characterized a series of transcription factors governing the differentiation of cranial neural crest-derived multipotent cells to the anterior and posterior palatal mesenchymal trajectories, respectively.Transcription factors Shox2 and Dlx1/2 exhibited top regulatory roles for the anterior and posterior palatal mesenchymal trajectories, respectively, showing significant enrichment in both motif accessibility and gene expression.

Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.

The etiology of cleft lip with or without cleft palate (CL/P), a common congenital birth defect, is complex, with genetic and epigenetic, as well as environmental, contributing factors. Recent studies suggest that fetal development is affected by maternal conditions through microRNAs (miRNAs), a group of short noncoding RNAs. Here, we show that miR-129-5p and miR-340-5p suppress cell proliferation in both primary mouse embryonic palatal mesenchymal cells and O9-1 cells, a neural crest cell line, through the regulation of Sox5 and Trp53 by miR-129-5p, and the regulation of Chd7, Fign and Tgfbr1 by miR-340-5p. Notably, miR-340-5p, but not miR-129-5p, was upregulated following all-trans retinoic acid (atRA; tretinoin) administration, and a miR-340-5p inhibitor rescued the cleft palate (CP) phenotype in 47% of atRA-induced CP mice. We have previously reported that a miR-124-3p inhibitor can also partially rescue the CP phenotype in atRA-induced CP mouse model. In this study, we found that a cocktail of miR-124-3p and miR-340-5p inhibitors rescued atRA-induced CP with almost complete penetrance. Taken together, our results suggest that normalization of pathological miRNA expression can be a preventive intervention for CP.

Mixing heterogeneous Li-ion conductive materials is one potential way to enhance Li-ion conductivity more than that of the parent materials. However, the huge number of possible compositions of par...

Cleft lip and palate is one of the most common congenital birth defects and has a complex etiology. Either genetic or environmental factors, or both, are involved at various degrees, and the type and severity of clefts vary. One of the longstanding questions is how environmental factors lead to craniofacial developmental anomalies. Recent studies highlight non-coding RNAs as potential epigenetic regulators in cleft lip and palate. In this review, we will discuss microRNAs, a type of small non-coding RNAs that can simultaneously regulate expression of many downstream target genes, as a causative mechanism of cleft lip and palate in humans and mice.

Sjögren's syndrome (SjS) is a chronic autoimmune disease characterized by immune cell infiltration of the exocrine glands, mainly the salivary and lacrimal glands. Despite recent advances in the clinical and mechanistic characterization of the disease, its etiology remains largely unknown. Here, we report that mice with a deficiency for either Atg7 or Atg3, which are enzymes involved in the ubiquitin modification pathway, in the salivary glands exhibit a SjS-like phenotype, characterized by immune cell infiltration with autoantibody detection, acinar cell death, and dry mouth. Prior to the onset of the SjS-like phenotype in these null mice, we detected an accumulation of secretory vesicles in the acinar cells of the salivary glands and found that GATE16, an uncharacterized autophagy-related molecule activated by ATG7 (E1-like enzyme) and ATG3 (E2-like enzyme), was highly expressed in these cells. Notably, GATE16 was activated by isoproterenol, an exocytosis inducer, and localized on the secretory vesicles in the acinar cells of the salivary glands. Failure to activate GATE16 was correlated with exocytosis defects in the acinar cells of the salivary glands in Atg7 and Atg3 cKO mice. Taken together, our results show that GATE16 activation regulated by the autophagic machinery is crucial for exocytosis and that defects in this pathway cause SjS.

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.

High-resolution computed tomography (CT) is widely used to assess bone structure under physiological and pathological conditions. Although the analytic protocols and parameters for micro-CT (μCT) analyses in mice are standardized for long bones, vertebrae, and the palms in aging mice, they have not yet been established for craniofacial bones. In this study, we conducted a morphometric assessment of craniofacial bones, in comparison with long bones, in aging mice. Although age-related changes were observed in the microarchitecture of the femur, tibia, vertebra, and basisphenoid bone, and were more pronounced in females than in males, the microarchitecture of both the interparietal bone and body of the mandible, which develop by intramembranous ossification, was less affected by age and sex. By contrast, the condyle of the mandible was more affected by aging in males compared to females. Taken together, our results indicate that mouse craniofacial bones are uniquely affected by age and sex.

The etiology of cleft lip with/without cleft palate (CL/P), one of the most frequent craniofacial birth defects worldwide, is complicated by contributions of both genetic and environmental factors. Understanding the etiology of these conditions is essential for developing preventive strategies. This study thus aims to identify regulatory networks of microRNAs (miRNAs), transcriptional factors (TFs) and non-TF genes associated with cleft lip (CL) that are conserved in humans and mice. Notably, we found that miR-27b, miR-133b, miR-205, miR-376b and miR-376c were involved in the regulation of CL-associated gene expression in both humans and mice. Among the candidate miRNAs, the overexpression of miR-27b, miR-133b and miR-205, but not miR-376b and miR-376c, significantly inhibited cell proliferation through suppression of CL-associated genes (miR-27b suppressed PAX9 and RARA; miR-133b suppressed FGFR1, PAX7, and SUMO1; and miR-205 suppressed PAX9 and RARA) in cultured human and mouse lip mesenchymal cells. Taken together, our results suggest that elevated expression of miR-27b, miR-133b and miR-205 may play a crucial role in CL through the suppression of genes associated with CL.

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.

It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an important effect on the proliferation and differentiation ability of MSCs. Utilizing a co-culture system comprising MSCs and HSCs, it is suggested that signaling from hematopoietic cells to MSCs supports cell proliferation. Interestingly, the enhanced proliferation ability of the HSCs was decreased in c-Mpl knock-out HSCs (c-Mpl-KO). In addition, the MSCs co-cultured with c-Mpl-KO HSCs had reduced MSC marker expression (PDGFRa and Sca-1) compared to the MSCs co-cultured with c-Mpl-wild-type HSCs. These results suggest that a hematopoietic–mesenchymal signal exists, and that the state of the HSCs is important for the stability of MSC properties.

Carbohydrates, fats, and proteins are the underlying energy sources for animals and are catabolized through specific biochemical cascades involving numerous enzymes. The catabolites and metabolites in these metabolic pathways are crucial for many cellular functions; therefore, an imbalance and/or dysregulation of these pathways causes cellular dysfunction, resulting in various metabolic diseases. Bone, a highly mineralized organ that serves as a skeleton of the body, undergoes continuous active turnover, which is required for the maintenance of healthy bony components through the deposition and resorption of bone matrix and minerals. This highly coordinated event is regulated throughout life by bone cells such as osteoblasts, osteoclasts, and osteocytes, and requires synchronized activities from different metabolic pathways. Here, we aim to provide a comprehensive review of the cellular metabolism involved in bone development and homeostasis, as revealed by mouse genetic studies.

Orofacial cleft (OFC) is one of the most prevalent birth defects, leading to substantial and long-term burdens in a newborn’s quality of life. Although studies revealed several genetic variants associated with the birth defect, novel approaches may provide additional clues about its etiology. Using the Center for Craniofacial and Dental Genetics project data (n = 10,542), we performed linear mixed-model analyses to study the genetic compositions of OFC and investigated the dependence among identified loci using conditional analyses. To identify genes associated with OFC, we conducted a transcriptome-wide association study (TWAS) based on predicted expression levels. In addition to confirming the previous findings at four loci, 1q32.2, 8q24, 2p24.2 and 17p13.1, we untwined two independent loci at 1q32.2, TRAF3IP3 and IRF6. The sentinel SNP in TRAF3IP3 (rs2235370, p-value = 5.15 × 10−9) was independent of the sentinel SNP at IRF6 (rs2235373, r2 &lt, 0.3). We found that the IRF6 effect became nonsignificant once the 8q24 effect was conditioned, while the TRAF3IP3 effect remained significant. Furthermore, we identified nine genes associated with OFC in TWAS, implicating a glutathione synthesis and drug detoxification pathway. We identified some meaningful additions to the OFC etiology using novel statistical methods in the existing data.

The mammalian salivary gland develops as a highly branched structure designed to produce and secrete saliva. This review focuses on research conducted on mammalian salivary gland development, particularly on the differentiation of acinar, ductal, and myoepithelial cells. We discuss recent studies that provide conceptual advances in the understanding of the molecular mechanisms of salivary gland development. In addition, we describe the organogenesis of submandibular glands (SMGs), model systems used for the study of SMG development, and the key signaling pathways as well as cellular processes involved in salivary gland development. The findings from the recent studies elucidating the identity of stem/progenitor cells in the SMGs, and the process by which they are directed along a series of cell fate decisions to form functional glands, are also discussed. Advances in genetic tools and tissue engineering strategies will significantly increase our knowledge about the mechanisms by which signaling pathways and cells establish tissue architecture and function during salivary gland development, which may also be conserved in the growth and development of other organ systems. An increased knowledge of organ development mechanisms will have profound implications in the design of therapies for the regrowth or repair of injured tissues. In addition, understanding how the processes of cell survival, expansion, specification, movement, and communication with neighboring cells are regulated under physiological and pathological conditions is critical to the development of future treatments.

Cleft lip (CL) with/without cleft palate (CP) (hereafter CL/P) is the second most common congenital birth defect, affecting 7.94 to 9.92 children per 10,000 live births worldwide, followed by Down syndrome. An increasing number of genes have been identified as affecting susceptibility and/or as causative genes for CL/P in mouse genetic and chemically-induced CL and CP studies, as well as in human genome-wide association studies and linkage analysis. While marked progress has been made in the identification of genetic and environmental risk factors for CL/P, the interplays between these factors are not yet fully understood. This review aims to summarize our current knowledge of CL and CP from genetically engineered mouse models and environmental factors that have been studied in mice. Understanding the regulatory mechanism(s) of craniofacial development may not only advance our understanding of craniofacial developmental biology, but could also provide approaches for the prevention of birth defects and for tissue engineering in craniofacial tissue regeneration.

Craniofacial development is regulated through dynamic and complex mechanisms that involve various signaling cascades and gene regulations. Disruption of such regulations can result in craniofacial birth defects. Here, we propose the first developmental stage-specific network approach by integrating two crucial regulators, transcription factors (TFs) and microRNAs (miRNAs), to study their co-regulation during craniofacial development. Specifically, we used TFs, miRNAs and non-TF genes to form feed-forward loops (FFLs) using genomic data covering mouse embryonic days E10.5 to E14.5. We identified key novel regulators (TFs Foxm1, Hif1a, Zbtb16, Myog, Myod1 and Tcf7, and miRNAs miR-340-5p and miR-129-5p) and target genes (Col1a1, Sgms2 and Slc8a3) expression of which changed in a developmental stage-dependent manner. We found that the Wnt-FoxO-Hippo pathway (from E10.5 to E11.5), tissue remodeling (from E12.5 to E13.5) and miR-129-5p-mediated Col1a1 regulation (from E10.5 to E14.5) might play crucial roles in craniofacial development. Enrichment analyses further suggested their functions. Our experiments validated the regulatory roles of miR-340-5p and Foxm1 in the Wnt-FoxO-Hippo subnetwork, as well as the role of miR-129-5p in the miR-129-5p–Col1a1 subnetwork. Thus, our study helps understand the comprehensive regulatory mechanisms for craniofacial development.

Background Cleft lip (CL) is one of the most common congenital birth defects with complex etiology. While genome-wide association studies (GWAS) have made significant advances in our understanding of mutations and their related genes with potential involvement in the etiology of CL, it remains unknown how these genes are functionally regulated and interact with each other in lip development. Currently, identifying the disease-causing genes in human CL is urgently needed. So far, the causative CL genes have been largely undiscovered, making it challenging to design experiments to validate the functional influence of the mutations identified from large genomic studies such as CL GWAS. Results Transcription factors (TFs) and microRNAs (miRNAs) are two important regulators in cellular system. In this study, we aimed to investigate the genetic interactions among TFs, miRNAs and the CL genes curated from the previous studies. We constructed miRNA-TF co-regulatory networks, from which the critical regulators as putative drivers in CL were examined. Based on the constructed networks, we identified ten critical hub genes with prior evidence in CL. Furthermore, the analysis of partitioned regulatory modules highlighted a number of biological processes involved in the pathology of CL, including a novel pathway “Signaling pathway regulating pluripotency of stem cells”. Our subnetwork analysis pinpointed two candidate miRNAs, hsa-mir-27b and hsa-mir-497, activating the Wnt pathway that was associated with CL. Our results were supported by an independent gene expression dataset in CL. Conclusions This study represents the first regulatory network analysis of CL genes. Our work presents a global view of the CL regulatory network and a novel approach on investigating critical miRNAs, TFs and genes via combinatory regulatory networks in craniofacial development. The top genes and miRNAs will be important candidates for future experimental validation of their functions in CL. Electronic supplementary material The online version of this article (10.1186/s12920-018-0458-3) contains supplementary material, which is available to authorized users.

Background Cleft lip with or without cleft palate (CL/P) is one of the most common congenital human birth defects. A combination of genetic and epidemiology studies has contributed to a better knowledge of CL/P-associated candidate genes and environmental risk factors. However, the etiology of CL/P remains not fully understood. In this study, to identify new CL/P-associated genes, we conducted an integrative analysis using our in-house network tools, dmGWAS [dense module search for Genome-Wide Association Studies (GWAS)] and EW_dmGWAS (Edge-Weighted dmGWAS), in a combination with GWAS data, the human protein-protein interaction (PPI) network, and differential gene expression profiles. Results A total of 87 genes were consistently detected in both European and Asian ancestries in dmGWAS. There were 31.0% (27/87) showed nominal significance with CL/P (gene-based p KIAA1598, GPR183, and ZMYND11 (p − 3). In EW_dmGWAS, we identified 253 and 245 module genes associated with CL/P for European ancestry and the Asian ancestry, respectively. Functional enrichment analysis demonstrated that these genes were involved in cell adhesion, protein localization to the plasma membrane, the regulation of the apoptotic signaling pathway, and other pathological conditions. A small proportion of genes (5.1% for European ancestry; 2.4% for Asian ancestry) had prior evidence in CL/P as annotated in CleftGeneDB database. Our analysis highlighted nine novel CL/P candidate genes (BRD1, CREBBP, CSK, DNM1L, LOR, PTPN18, SND1, TGS1, and VIM) and 17 previously reported genes in the top modules. Conclusions The genes identified through superimposing GWAS signals and differential gene expression profiles onto human PPI network, as well as their functional features, helped our understanding of the etiology of CL/P. Our multi-omics integrative analyses revealed nine novel candidate genes involved in CL/P.

This article presents data on genes associated with cleft palate (CP), retrieved through both a full-text systematic review and a mouse genome informatics (MGI) database search. In order to group CP-associated genes according to function, pathway, biological process, and cellular component, the genes were analyzed using category enrichment bioinformatics tools, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). This approach provides invaluable opportunities for the identification of candidate pathways and genes in CP research.

Cleft palate (CP) is the most prevalent craniofacial deformity, with ethnic and geographic variation in prevalence in humans. Mice have been used as an animal model to study the cause(s) of CP by several approaches, including genetic and chemical-induced approaches. Mouse genetic approaches revealed that significant amounts of genes are involved in the CP pathology. The aim of this study was to identify common features of CP-associated genes and to explore the roles of microRNAs (miRNAs) as important post-transcriptional regulators that may be involved in the regulation of CP genes. To generate an accurate list of genes associated with CP, we first conducted systematic literature searches through main databases such as Medline, Embase, and PubMed, as well as other sources such as Scopus and Mouse Genome Informatics. We found that 195 mouse strains with single-gene mutations and 140 mouse strains with compound-gene mutations were reported to have CP. The CP genes were categorized by functions and pathways using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology annotations, highlighting the contribution of cellular metabolism to CP. A total of 18 miRNAs were involved in the regulation of multiple CP genes. Human genotype-phenotype analysis revealed that variants in five human homologous CP genes (IRF6, FOXE1, VAX1, WNT9B, and GAD1) significantly contributed to the human CP phenotype. Thus, our results suggest that cellular metabolism and miRNAs play an important role in the regulation of genetic pathways and networks crucial for palatal formation.

Human linkage studies suggest that craniofacial deformities result from either genetic mutations related to cholesterol metabolism or high-cholesterol maternal diets. However, little is known about the precise roles of intracellular cholesterol metabolism in the development of craniofacial bones, the majority of which are formed through intramembranous ossification. Here, we show that an altered cholesterol metabolic status results in abnormal osteogenesis through dysregulation of primary cilium formation during bone formation. We found that cholesterol metabolic aberrations, induced through disruption of either Dhcr7 (which encodes an enzyme involved in cholesterol synthesis) or Insig1 and Insig2 (which provide a negative feedback mechanism for cholesterol biosynthesis), result in osteoblast differentiation abnormalities. Notably, the primary cilia responsible for sensing extracellular cues were altered in number and length through dysregulated ciliary vesicle fusion in Dhcr7 and Insig1/2 mutant osteoblasts. As a consequence, WNT/β-catenin and hedgehog signaling activities were altered through dysregulated primary cilium formation. Strikingly, the normalization of defective cholesterol metabolism by simvastatin, a drug used in the treatment of cholesterol metabolic aberrations, rescued the abnormalities in both ciliogenesis and osteogenesis in vitro and in vivo. Thus, our results indicate that proper intracellular cholesterol status is crucial for primary cilium formation during skull formation and homeostasis.

Craniofacial development is regulated through dynamic and complex mechanisms that involve various signaling cascades and gene regulations. Disruption of such regulations may result in craniofacial birth defects. Here, we propose the first developmental stage-specific network approach by integrating two critical regulators, transcription factor (TF) and microRNA (miRNA), to study their co-regulation during craniofacial development. Specifically, we used TFs, miRNAs, and non-TF genes to form Feed-forward Loops (FFLs) using genomic data covering mouse embryonic days E10.5 to E14.5. We identified key novel regulators (TFs: Foxm1, Hif1a, Zbtb16, Myog, Myod1, and Tcf7, and miRNAs: miR-340-5p and miR-129-5p) and target genes (Col1a1, Sgms2, and Slc8a3) whose expression changed in a developmental stage-dependent manner. We found Wnt-FoxO-Hippo pathway (from E10.5 to E11.5), tissue remodeling (from E12.5 to E13.5), and miR-129-5p-mediated Col1a1 regulation (from E10.5 to E14.5) might play crucial roles in craniofacial development. Enrichment analyses further suggested their functions. Our experiments validated the regulatory roles of miR-340-5p and Foxm1 in Wnt-FoxO-Hippo subnetwork, as well as the role of miR-129-5p in the miR-129-5p-Col1a1 subnetwork. Thus, our study helps understand comprehensive regulatory mechanisms for craniofacial development.

BackgroundCleft lip (CL), one of the most common congenital birth defects, shows considerable geographic and ethnic variation, with contribution of both genetic and environmental factors. Mouse genetic studies have identified several CL-associated genes. However, it remains elusive how these CL-associated genes are regulated and involved in CL. Environmental factors may regulate these genes at the post-transcriptional level through the regulation of non-coding microRNAs (miRNAs). In this study, we sought to identify miRNAs associated with CL in mice.ResultsThrough a systematic literature review and a Mouse Genome Informatics (MGI) database search, we identified 55 genes that were associated with CL in mice. Subsequent bioinformatic analysis of these genes predicted that a total of 33 miRNAs target multiple CL-associated genes, with 20 CL-associated genes being potentially regulated by multiple miRNAs. To experimentally validate miRNA function in cell proliferation, we conducted cell proliferation/viability assays for the selected five candidate miRNAs (miR-124-3p, let-7a-5p, let-7b-5p, let-7c-5p, and let-7d-5p). Overexpression of miR-124-3p, but not of the others, inhibited cell proliferation through suppression of CL-associated genes in cultured mouse embryonic lip mesenchymal cells (MELM cells) isolated from the developing mouse lip region. By contrast, miR-124-3p knockdown had no effect on MELM cell proliferation. This miRNA-gene regulatory mechanism was mostly conserved in O9–1 cells, an established cranial neural crest cell line. Expression of miR-124-3p was low in the maxillary processes at E10.5, when lip mesenchymal cells proliferate, whereas it was greatly increased at later developmental stages, suggesting that miR-124-3p expression is suppressed during the proliferation phase in normal palate development.ConclusionsOur findings indicate that upregulated miR-124-3p inhibits cell proliferation in cultured lip cells through suppression of CL-associated genes. These results will have a significant impact, not only on our knowledge about lip morphogenesis, but also on the development of clinical approaches for the diagnosis and prevention of CL.

Cleft palate (CP) is the second most common congenital birth defect. The etiology of CP is complicated, with involvement of various genetic and environmental factors. To investigate the gene regulatory mechanisms, we designed a powerful regulatory analytical approach to identify the conserved regulatory networks in humans and mice, from which we identified critical microRNAs (miRNAs), target genes and regulatory motifs (miRNA–TF–gene) related to CP. Using our manually curated genes and miRNAs with evidence in CP in humans and mice, we constructed miRNA and transcription factor (TF) co-regulation networks for both humans and mice. A consensus regulatory loop (miR17/miR20a–FOXE1–PDGFRA) and eight miRNAs (miR-140, miR-17, miR-18a, miR-19a, miR-19b, miR-20a, miR-451a and miR-92a) were discovered in both humans and mice. The role of miR-140, which had the strongest association with CP, was investigated in both human and mouse palate cells. The overexpression of miR-140-5p, but not miR-140-3p, significantly inhibited cell proliferation. We further examined whether miR-140 overexpression could suppress the expression of its predicted target genes (BMP2, FGF9, PAX9 and PDGFRA). Our results indicated that miR-140-5p overexpression suppressed the expression of BMP2 and FGF9 in cultured human palate cells and Fgf9 and Pdgfra in cultured mouse palate cells. In summary, our conserved miRNA–TF–gene regulatory network approach is effective in detecting consensus miRNAs, motifs, and regulatory mechanisms in human and mouse CP.

Background Cleft palate (CP) is the second most common congenital birth defect; however, the relationship between CP-associated genes and epigenetic regulation remains largely unknown. In this study, we investigated the contribution of microRNAs (miRNAs) to cell proliferation and regulation of genes involved in CP development. Methods In order to identify all genes for which mutations or association/linkage have been found in individuals with CP, we conducted a systematic literature search, followed by bioinformatics analyses for these genes. We validated the bioinformatics results experimentally by conducting cell proliferation assays and miRNA-gene regulatory analyses in cultured human palatal mesenchymal cells treated with each miRNA mimic. Results We identified 131 CP-associated genes in the systematic review. The bioinformatics analysis indicated that the CP genes were associated with signaling pathways, microRNAs (miRNAs), metabolic pathways, and cell proliferation. A total 17 miRNAs were recognized as potential modifiers of human CP genes. To validate miRNA function in cell proliferation, a main cause of CP, we conducted cell proliferation/viability assays for the top 11 candidate miRNAs from our bioinformatics analysis. Overexpression of miR-133b, miR-374a-5p, and miR-4680-3p resulted in a more than 30% reduction in cell proliferation activity in human palatal mesenchymal cell cultures. We found that several downstream target CP genes predicted by the bioinformatics analyses were significantly downregulated through induction of these miRNAs (FGFR1, GCH1, PAX7, SMC2, and SUMO1 by miR-133b; ARNT, BMP2, CRISPLD1, FGFR2, JARID2, MSX1, NOG, RHPN2, RUNX2, WNT5A and ZNF236 by miR-374a-5p; and ERBB2, JADE1, MTHFD1 and WNT5A by miR-4680-3p) in cultured cells. Conclusions Our results indicate that miR-374a-5p, miR-4680-3p, and miR-133b regulate expression of genes that are involved in the etiology of human CP, providing insight into the association between CP-associated genes and potential targets of miRNAs in palate development. Electronic supplementary material The online version of this article (10.1186/s12920-019-0546-z) contains supplementary material, which is available to authorized users.

Background The etiology of cleft lip with or without cleft palate (CL/P), a common congenital birth defect, is complex and involves the contribution of genetic and environmental factors. Although many candidate genes have been identified, the regulation and interaction of these genes in CL/P remain unclear. In addition, the contribution of microRNAs (miRNAs), non-coding RNAs that regulate the expression of multiple genes, to the etiology of CL/P is largely unknown. Methods To identify the signatures of causative biological pathways for human CL/P, we conducted a systematic literature review for human CL/P candidate genes and subsequent bioinformatics analyses. Functional enrichment analyses of the candidate CL/P genes were conducted using the pathway databases GO and KEGG. The miRNA-mediated post-transcriptional regulation of the CL/P candidate genes was analyzed with miRanda, PITA, and TargetScan, and miRTarbase. Genotype-phenotype association analysis was conducted using GWAS. The functional significance of the candidate miRNAs was evaluated experimentally in cell proliferation and target gene regulation assays in human lip fibroblasts. Results Through an extensive search of the main biomedical databases, we mined 177 genes with mutations or association/linkage reported in individuals with CL/P, and considered them as candidate genes for human CL/P. The genotype-phenotype association study revealed that mutations in 12 genes (ABCA4, ADAM3A, FOXE1, IRF6, MSX2, MTHFR, NTN1, PAX7, TP63, TPM1, VAX1, and WNT9B) were significantly associated with CL/P. In addition, our bioinformatics analysis predicted 16 microRNAs (miRNAs) to be post-transcriptional regulators of CL/P genes. To validate the bioinformatics results, the top six candidate miRNAs (miR-124-3p, miR-369-3p, miR-374a-5p, miR-374b-5p, miR-497-5p, and miR-655-3p) were evaluated by cell proliferation/survival assays and miRNA-gene regulation assays in cultured human lip fibroblasts. We found that miR-497-5p and miR-655-3p significantly suppressed cell proliferation in these cells. Furthermore, the expression of the predicted miRNA-target genes was significantly downregulated by either miR-497-5p or miR-655-3p mimic. Conclusion Expression of miR-497-5p and miR-655-3p suppresses cell proliferation through the regulation of human CL/P-candidate genes. This study provides insights into the role of miRNAs in the etiology of CL/P and suggests possible strategies for the diagnosis of CL/P. Electronic supplementary material The online version of this article (10.1186/s12920-019-0535-2) contains supplementary material, which is available to authorized users.

It has been long appreciated that sex-hormone receptors are expressed in various non-gonadal organs. However, it remains unclear how sex hormones regulate the morphogenesis of these non-gonadal organs. To address this question, we used a male mouse model of androgen-dependent salivary gland morphogenesis. Mice with excessive cholesterol synthesis in the salivary glands exhibited defects in the maturation of granular convoluted tubules (GCTs), which is regulated through sex hormone-dependent cascades. We found that excessive cholesterol synthesis resulted in autophagy failure specifically in the duct cells of salivary glands, followed by the accumulation of NRF2, a transcription factor known as one of the specific substrates for autophagy. The accumulated NRF2 suppressed the expression of Foxa1, which forms a transcriptional complex with the androgen receptor to regulate target genes. Taken together, our results indicate that cholesterol metabolism plays a crucial role in GCT differentiation through autophagy.

Bone is an active organ that is continuously remodeled throughout life via formation and resorption; therefore, a fine-tuned bone (re)modeling is crucial for bone homeostasis and is closely connected with energy metabolism. Amino acids are essential for various cellular functions as well as an energy source, and their synthesis and catabolism (e.g., metabolism of carbohydrates and fatty acids) are regulated through numerous enzymatic cascades. In addition, the intracellular levels of amino acids are maintained by autophagy, a cellular recycling system for proteins and organelles; under nutrient deprivation conditions, autophagy is strongly induced to compensate for cellular demands and to restore the amino acid pool. Metabolites derived from amino acids are known to be precursors of bioactive molecules such as second messengers and neurotransmitters, which control various cellular processes, including cell proliferation, differentiation, and homeostasis. Thus, amino acid metabolism and autophagy are tightly and reciprocally regulated in our bodies. This review discusses the current knowledge and potential links between bone diseases and deficiencies in amino acid metabolism and autophagy.