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.

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.

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.

Role of Metabolism in Bone Development and Homeostasis.

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.

DNA methylation of the Klf14 gene region in whole blood cells provides prediction for the chronic inflammation in the adipose tissue.

Krüppel-Like Factor 14 (KLF14) gene, which appears to be a master regulator of gene expression in the adipose tissue and have previously been associated with BMI and Type 2 diabetes (T2D) by large genome-wide association studies. In order to find predictive biomarkers for the development of T2D, it is necessary to take epigenomic changes affected by environmental factors into account. This study focuses on ageing and obesity, which are T2D risk factors, and examines epigenetic changes and inflammatory changes. We investigated DNA methylation changes in the Klf14 promoter region in different organs of mice for comparing aging and weight. We found that methylation levels of these sites were increased with aging and weight in the spleen, the adipose tissue, the kidney, the lung, the colon and the whole blood cells. In addition, in the spleen, the adipose tissue and the whole blood, these epigenetic changes were also significantly associated with inflammatory levels. Moreover, not only Klf14, but also expression levels of some downstream genes were decreased with methylation in the spleen, the adipose tissue and the whole blood cells. Taken together, our results suggest that methylation changes of Klf14 in those tissues may be associated with changes in gene expression and inflammation on the adipose tissue of obesity and T2D. In addition, the methylation changes in the whole blood cells may serve as a predictive epigenetic biomarker for the development of T2D.

Serum miR-375-3p increase in mice exposed to a high dose of ionizing radiation.

Exposure to high-doses of ionizing radiation (IR) leads to development of a strong acute radiation syndrome (ARS) in mammals. ARS manifests after a latency period and it is important to develop fast prognostic biomarkers for its early detection and assessment. Analysis of chromosomal aberrations in peripheral blood lymphocytes is the gold standard of biological dosimetry, but it fails after high doses of IR. Therefore, it is important to establish novel biomarkers of exposure that are fast and reliable also in the high dose range. Here, we investigated the applicability of miRNA levels in mouse serum. We found significantly increased levels of miR-375-3p following whole body exposure to 7 Gy of X-rays. In addition, we analyzed their levels in various organs of control mice and found them to be especially abundant in the pancreas and the intestine. Following a dose of 7 Gy, extensive cell death occurred in these tissues and this correlated negatively with the levels of miR-375-3p in the organs. We conclude that high expressing tissues of miR-375-3p may secrete this miRNA in serum following exposure to 7 Gy. Therefore, elevated miR-375-3p in serum may be a predictor of tissue damage induced by exposure to a high radiation dose.

Genome-wide DNA methylation analysis reveals hypomethylation in the low-CpG promoter regions in lymphoblastoid cell lines.

BACKGROUND: Epidemiological studies of DNA methylation profiles may uncover the molecular mechanisms through which genetic and environmental factors contribute to the risk of multifactorial diseases. There are two types of commonly used DNA bioresources, peripheral blood cells (PBCs) and EBV-transformed lymphoblastoid cell lines (LCLs), which are available for genetic epidemiological studies. Therefore, to extend our knowledge of the difference in DNA methylation status between LCLs and PBCs is important in human population studies that use these DNA sources to elucidate the epigenetic risks for multifactorial diseases. We analyzed the methylation status of the autosomes for 192 and 92 DNA samples that were obtained from PBCs and LCLs, respectively, using a human methylation 450 K array. After excluding SNP-associated methylation sites and low-call sites, 400,240 sites were subjected to analysis using a generalized linear model with cell type, sex, and age as the independent variables. RESULTS: We found that the large proportion of sites showed lower methylation levels in LCLs compared with PBCs, which is consistent with previous reports. We also found that significantly different methylation sites tend to be located on the outside of the CpG island and in a region relatively far from the transcription start site. Additionally, we observed that the methylation change of the sites in the low-CpG promoter region was remarkable. Finally, it was shown that the correlation between the chronological age and ageing-associated methylation sites in ELOVL2 and FHL2 in the LCLs was weaker than that in the PBCs. CONCLUSIONS: The methylation levels of highly methylated sites of the low-CpG-density promoters in PBCs decreased in the LCLs, suggesting that the methylation sites located in low-CpG-density promoters could be sensitive to demethylation in LCLs. Despite being generated from a single cell type, LCLs may not always be a proxy for DNA from PBCs in studies of epigenome-wide analysis attempting to elucidate the role of epigenetic change in disease risks.