Craniofacial bone anomalies related to cholesterol synthesis defects.

DHCR7 and SC5D are enzymes crucial for cholesterol biosynthesis, and mutations in their genes are associated with developmental disorders, which are characterized by craniofacial deformities. We have recently reported that a loss of either Dhcr7 or Sc5d results in a failure in osteoblast differentiation. However, it remains unclear to what extent a loss of function in either DHCR7 or SC5D affects craniofacial skeletal formation. Here, using micro computed tomography (µCT), we found that the bone phenotype differs in Dhcr7-/- and Sc5d-/- mice in a location-specific fashion. For instance, in Sc5d-/- mice, although craniofacial bones were overall affected, some bone segments, such as the anterior part of the premaxilla, the anterior-posterior length of the frontal bone, and the main body of the mandible, did not present significant differences compared to WT controls. By contrast, in Dhcr7-/- mice, while craniofacial bones were not much affected, the frontal bone was larger in width and volume, and the maxilla and palatine bone were hypoplastic, compared to WT controls. Interestingly the mandible in Dhcr7-/- mice was mainly affected at the condylar region, not the body. Thus, these results help us understand which bones and how greatly they are affected by cholesterol metabolism aberrations in Dhcr7-/- and Sc5d-/- mice.

Single-cell multiomics decodes regulatory programs for mouse secondary palate development.

Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile 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. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.

Loss of Sc5d results in micrognathia due to a failure in osteoblast differentiation.

INTRODUCTION: Mutations in genes related to cholesterol metabolism, or maternal diet and health status, affect craniofacial bone formation. However, the precise role of intracellular cholesterol metabolism in craniofacial bone development remains unclear. OBJECTIVE: The aim of this study is to determine how cholesterol metabolism aberrations affect craniofacial bone development. METHODS: Mice with a deficiency in Sc5d, which encodes an enzyme involved in cholesterol synthesis, were analyzed with histology, micro computed tomography (microCT), and cellular and molecular biological methods. RESULTS: Sc5d null mice exhibited mandible hypoplasia resulting from defects in osteoblast differentiation. The activation of the hedgehog and WNT/ß-catenin signaling pathways, which induce expression of osteogenic genes Col1a1 and Spp1, was compromised in the mandible of Sc5d null mice due to a failure in the formation of the primary cilium, a cell surface structure that senses extracellular cues. Treatments with an inducer of hedgehog or WNT/ß-catenin signaling or with simvastatin, a drug that restores abnormal cholesterol production, partially rescued the defects in osteoblast differentiation seen in Sc5d mutant cells. CONCLUSION: Our results indicate that loss of Sc5d results in mandibular hypoplasia through defective primary cilia-mediated hedgehog and WNT/ß-catenin signaling pathways.

MicroRNAs and Gene Regulatory Networks Related to Cleft Lip and Palate.

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.

Hematopoietic-Mesenchymal Signals Regulate the Properties of Mesenchymal Stem Cells.

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.

Impaired GATE16-mediated exocytosis in exocrine tissues causes Sjögren's syndrome-like exocrinopathy.

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.

Micro-computed tomography assessment of bone structure in aging 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.

Crucial Roles of microRNA-16-5p and microRNA-27b-3p in Ameloblast Differentiation Through Regulation of Genes Associated With Amelogenesis Imperfecta.

Amelogenesis imperfecta is a congenital disorder within a heterogeneous group of conditions characterized by enamel hypoplasia. Patients suffer from early tooth loss, social embarrassment, eating difficulties, and pain due to an abnormally thin, soft, fragile, and discolored enamel with poor aesthetics and functionality. The etiology of amelogenesis imperfecta is complicated by genetic interactions. To identify mouse amelogenesis imperfecta-related genes (mAIGenes) and their respective phenotypes, we conducted a systematic literature review and database search and found and curated 70 mAIGenes across all of the databases. Our pathway enrichment analysis indicated that these genes were enriched in tooth development-associated pathways, forming four distinct groups. To explore how these genes are regulated and affect the phenotype, we predicted microRNA (miRNA)-gene interaction pairs using our bioinformatics pipeline. Our miRNA regulatory network analysis pinpointed that miR-16-5p, miR-27b-3p, and miR-23a/b-3p were hub miRNAs. The function of these hub miRNAs was evaluated through ameloblast differentiation assays with/without the candidate miRNA mimics using cultured mouse ameloblast cells. Our results revealed that overexpression of miR-16-5p and miR-27b-3p, but not miR-23a/b-3p, significantly inhibited ameloblast differentiation through regulation of mAIGenes. Thus, our study shows that miR-16-5p and miR-27b-3p are candidate pathogenic miRNAs for amelogenesis imperfecta.

Suppression of microRNA 124-3p and microRNA 340-5p ameliorates retinoic acid-induced cleft palate in mice.

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.

Dexamethasone Suppresses Palatal Cell Proliferation through miR-130a-3p.

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.

Mesenchymal stromal cells in the bone marrow niche consist of multi-populations with distinct transcriptional and epigenetic properties.

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.

Identification of microRNAs and gene regulatory networks in cleft lip common in humans and mice.

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.

MicroRNA-124-3p Plays a Crucial Role in Cleft Palate Induced by Retinoic Acid.

Cleft lip with/without cleft palate (CL/P) is one of the most common congenital birth defects, showing the complexity of both genetic and environmental contributions [e.g., maternal exposure to alcohol, cigarette, and retinoic acid (RA)] in humans. Recent studies suggest that epigenetic factors, including microRNAs (miRs), are altered by various environmental factors. In this study, to investigate whether and how miRs are involved in cleft palate (CP) induced by excessive intake of all-trans RA (atRA), we evaluated top 10 candidate miRs, which were selected through our bioinformatic analyses, in mouse embryonic palatal mesenchymal (MEPM) cells as well as in mouse embryos treated with atRA. Among them, overexpression of miR-27a-3p, miR-27b-3p, and miR-124-3p resulted in the significant reduction of cell proliferation in MEPM cells through the downregulation of CP-associated genes. Notably, we found that excessive atRA upregulated the expression of miR-124-3p, but not of miR-27a-3p and miR-27b-3p, in both in vivo and in vitro. Importantly, treatment with a specific inhibitor for miR-124-3p restored decreased cell proliferation through the normalization of target gene expression in atRA-treated MEPM cells and atRA-exposed mouse embryos, resulting in the rescue of CP in mice. Taken together, our results indicate that atRA causes CP through the induction of miR-124-3p in mice.

Overexpression of miR-1306-5p, miR-3195, and miR-3914 Inhibits Ameloblast Differentiation through Suppression of Genes Associated with Human Amelogenesis Imperfecta.

Amelogenesis imperfecta is a congenital form of enamel hypoplasia. Although a number of genetic mutations have been reported in humans, the regulatory network of these genes remains mostly unclear. To identify signatures of biological pathways in amelogenesis imperfecta, we conducted bioinformatic analyses on genes associated with the condition in humans. Through an extensive search of the main biomedical databases, we found 56 genes in which mutations and/or association/linkage were reported in individuals with amelogenesis imperfecta. These candidate genes were further grouped by function, pathway, protein-protein interaction, and tissue-specific expression patterns using various bioinformatic tools. The bioinformatic analyses highlighted a group of genes essential for extracellular matrix formation. Furthermore, advanced bioinformatic analyses for microRNAs (miRNAs), which are short non-coding RNAs that suppress target genes at the post-transcriptional level, predicted 37 candidates that may be involved in amelogenesis imperfecta. To validate the miRNA-gene regulation association, we analyzed the target gene expression of the top seven candidate miRNAs: miR-3195, miR-382-5p, miR-1306-5p, miR-4683, miR-6716-3p, miR-3914, and miR-3935. Among them, miR-1306-5p, miR-3195, and miR-3914 were confirmed to regulate ameloblast differentiation through the regulation of genes associated with amelogenesis imperfecta in AM-1 cells, a human ameloblastoma cell line. Taken together, our study suggests a potential role for miRNAs in amelogenesis imperfecta.

Excessive All-Trans Retinoic Acid Inhibits Cell Proliferation Through Upregulated MicroRNA-4680-3p in Cultured Human Palate Cells.

Cleft palate is the second most common congenital birth defect, and both environmental and genetic factors are involved in the etiology of the disease. However, it remains largely unknown how environmental factors affect palate development. Our previous studies show that several microRNAs (miRs) suppress the expression of genes involved in cleft palate. Here we show that miR-4680-3p plays a crucial role in cleft palate pathogenesis. We found that all-trans retinoic acid (atRA) specifically induces miR-4680-3p in cultured human embryonic palatal mesenchymal (HEPM) cells. Overexpression of miR-4680-3p inhibited cell proliferation in a dose-dependent manner through the suppression of expression of ERBB2 and JADE1, which are known cleft palate-related genes. Importantly, a miR-4680-3p-specific inhibitor normalized cell proliferation and altered expression of ERBB2 and JADE1 in cells treated with atRA. Taken together, our results suggest that upregulation of miR-4680-3p induced by atRA may cause cleft palate through suppression of ERBB2 and JADE1. Thus, miRs may be potential targets for the prevention and diagnosis of cleft palate.

Phenytoin Inhibits Cell Proliferation through microRNA-196a-5p in Mouse Lip Mesenchymal Cells.

Cleft lip (CL) is one of the most common birth defects. It is caused by either genetic mutations or environmental factors. Recent studies suggest that environmental factors influence the expression of noncoding RNAs [e.g., microRNA (miRNA)], which can regulate the expression of genes crucial for cellular functions. In this study, we examined which miRNAs are associated with CL. Among 10 candidate miRNAs (miR-98-3p, miR-101a-3p, miR-101b-3p, miR-141-3p, miR-144-3p, miR-181a-5p, miR-196a-5p, miR-196b-5p, miR-200a-3p, and miR-710) identified through our bioinformatic analysis of CL-associated genes, overexpression of miR-181a-5p, miR-196a-5p, miR-196b-5p, and miR-710 inhibited cell proliferation through suppression of genes associated with CL in cultured mouse embryonic lip mesenchymal cells (MELM cells) and O9-1 cells, a mouse cranial neural crest cell line. In addition, we found that phenytoin, an inducer of CL, decreased cell proliferation through miR-196a-5p induction. Notably, treatment with a specific inhibitor for miR-196a-5p restored cell proliferation through normalization of expression of CL-associated genes in the cells treated with phenytoin. Taken together, our results suggest that phenytoin induces CL through miR-196a-5p induction, which suppresses the expression of CL-associated genes.

Amino acid metabolism and autophagy in skeletal development and homeostasis.

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.