IntroductionSjögren’s disease (SjD) is a chronic autoimmune disorder characterized by dry mouth (xerostomia) and dry eyes (xerophthalmia) due to inflammation in exocrine glands, particularly the salivary and lacrimal glands. The condition presents a wide variety of clinical features, suggesting that it may involve heterogeneous conditions without a clear boundary. Although genome-wide association studies (GWAS) have identified several genetic variants associated with SjD, their roles in SjD pathogenesis remain unclear.MethodsIn this study, we aimed to identify single-nucleotide polymorphisms (SNPs) associated with SjD by categorizing patients based on four diagnostic markers: anti-Ro/SSA and anti-La/SSB antibodies, IgG levels, and lymphocyte foci, using a genotype-phenotype dataset (NCBI dbGaP phs000672.v1.p1) which included 594 SjD patients, 1,264 sicca symptomatic individuals without SjD diagnosis (as a control group), and 41 healthy individuals (as another control group) .Results and DiscussionWe identified SNPs associated with each subtype of SjD, organized by two factors of diagnostic markers, X (anti-SSA and/or anti-SSB autoantibody) and Y (IgG or lymphocyte foci), with an adjusted p-value less than 5x10-8. The SjD subtypes were classified as follows: Group A (Factors X+ and Y+), Group B (X+ and Y-), Group C (X- and Y+), and Group D (X- and Y-). We found distinct SNPs associated with each group of SjD patients. This study can help advance the SjD subtype investigation, supporting precision diagnosis and treatment of SjD.

Craniosynostosis (CS) is a congenital birth defect defined by the premature closure of one or more cranial sutures. Approximately 70% of CS cases are nonsyndromic, with underlying causes frequently remaining unidentified. This study seeks to identify short noncoding RNAs, specifically microRNAs (miRNAs), associated with syndromic, nonsyndromic, and all forms of CS to advance understanding of its etiology. Through a systematic review, a total of 165 genes were identified as associated with human CS (112 syndromic, 37 nonsyndromic, and 13 overlapping). Bioinformatic analyses identified several miRNAs capable of regulating these CS-related genes. We found that miR-377 and miR-335-3p were specifically involved in the regulation of genes associated with syndromic CS, while miR-371-5p, miR-329, and miR-204-5p were specifically involved in gene regulation related to nonsyndromic CS. In contrast, miR-651-3p, miR-362-3p, and miR-425 play a role in both syndromic and nonsyndromic CS. Subsequent enrichment analysis using ShinyGO revealed that the predicted targets of these nine candidate miRNAs were preferentially enriched in the TGF-beta signaling pathway. Notably, functional modulation of these miRNAs altered the undifferentiated state, cell proliferation, and osteogenic differentiation of suture progenitor cells. Taken together, our study indicates that these miRNAs play a role in CS by changing the cell characteristics of suture progenitor cells.

Midfacial hypoplasia is a developmental defect caused by insufficient growth of the nasal placodes and maxillary prominences. While genetic studies in mice have identified key genes involved in midfacial development, the regulation of these genes during craniofacial development remains poorly understood. In this study, we demonstrate that microRNAs, short non-coding RNAs, play a crucial role in regulating genes involved in midfacial development. We identified 224 genes associated with midfacial malformations in mice. Through bioinformatics analyses, we predicted that several microRNAs, specifically miR-129-5p, miR-381-3p, miR-124-3p, miR-136-5p, miR-448-3p, miR-374, miR-96, and miR-882, could regulate the expression of these genes. Among these, we experimentally focused on the top four candidate microRNAs: miR-129-5p, miR-381-3p, miR-124-3p, and miR-136-5p. Our findings revealed that the overexpression of these microRNAs inhibited cell proliferation and osteogenesis in nasal process mesenchymal cells. These microRNAs regulated genes associated with midfacial malformations in a dose-dependent manner. Taken together, our results emphasize the significance of pathogenic microRNA-gene networks in the cause of midfacial malformations.

Midline facial clefts are severe craniofacial defects that occur due to an underdeveloped frontonasal process. While genetic studies in mice have identified several genes that are crucial for midfacial development, the interactions and regulatory mechanisms of these genes during development remain unclear. In this study, we conducted a systematic review and database search to curate genes associated with midline facial clefts in mice. We identified a total of 78 relevant genes, which included 69 single-gene mutant mice, nine spontaneous models, and 20 compound mutant mice. We then performed bioinformatic analyses with these genes to identify candidate microRNAs (miRNAs) that may regulate the expression of genes related to midline facial clefts. Furthermore, we experimentally evaluated the four highest-ranking candidates—miR-320-3p, miR-381-3p, miR-27a-3p, and miR-124-3p—in O9-1 cells. Our results indicated that overexpression of any of these miRNAs inhibited cell proliferation through the suppression of genes associated with midline facial clefts. Thus, our results suggest that miR-320-3p, miR-381-3p, miR-27a-3p, and miR-124-3p are involved in the cause of midline facial anomalies.

Hypertelorism, characterized by an abnormal increase in the distance between the eyes, is often associated with various congenital birth defects. While there is increasing evidence suggesting common underlying mechanisms for hypertelorism, the role of microRNAs (miRNAs)—short noncoding RNAs that suppress target genes by inhibiting translation and degrading mRNA—in the condition’s pathogenesis remains unclear. This study aimed to identify the miRNAs associated with hypertelorism in mice. By searching the Mouse Genome Informatics (MGI) database and reviewing full-text references, we identified a total of 31 genes potentially related to hypertelorism. Advanced bioinformatics analyses revealed nine miRNAs that may regulate these genes. We experimentally evaluated candidate miRNAs in assays of cell proliferation and target gene regulation in primary cells isolated from developing frontonasal process mouse embryonic frontonasal mesenchymal and O9-1 cells, a murine neural crest cell line. Our findings indicated that overexpression of either miR-383-3p or miR-6951-3p stimulated cell proliferation, whereas miR-7116-3p and miR-124-3p did not have this effect. Additionally, we confirmed that miR-383-3p and miR-6951-3p regulated the expression of a set of hypertelorism-related genes in a dose-dependent manner. These results suggest that miR-383-3p and miR-6951-3p play significant roles in the development of hypertelorism.

Any failure in frontonasal development can lead to malformations at the middle facial region, such as frontonasal dysplasia, midfacial clefts, and hyper/hypotelorism. Various environmental factors influence morphogenesis through epigenetic regulations, including the action of noncoding microRNAs (miRNAs). However, it remains unclear how miRNAs are involved in the frontonasal development. In our analysis of publicly available miRNA-seq and RNA-seq datasets, we found that miR-28a-5p, miR-302a-3p, miR-302b-3p, and miR-302d-3p were differentially expressed in the frontonasal process during embryonic days 10.5 to 13.5 (E10.5-E13.5) in mice. Overexpression of these miRNAs led to a suppression of cell proliferation in cultured mouse embryonic frontonasal mesenchymal (MEFM) cells as well as in O9-1 cells, a cranial neural crest cell line. Through advanced bioinformatic analyses and miRNA-gene regulation assays, we identified that miR-28a-5p regulated a total of 25 genes, miR-302a-3p regulated 23 genes, miR-302b-3p regulated 22 genes, and miR-302d-3p regulated 20 genes. Notably, the expression of miR-302a/b/d-3p-unlike miR-28a-5p-was significantly upregulated by excessive exposure to all-trans retinoic acid (atRA) that induces craniofacial malformations. Inhibition of these miRNAs restored the reduced cell proliferation caused by atRA by normalizing the expression of target genes associated with frontonasal anomalies. Therefore, our findings suggest that miR-302a/b/d-3p plays a crucial role in the development of frontonasal malformations.

Orofacial clefts (OFC) are common congenital birth defects with various etiologies, including genetic variants. Online Mendelian Inheritance in Man (OMIM) annotated several hundred genes involving OFC. Furthermore, several hundreds of de novo variants (DNVs) have been identified from OFC individuals. Some DNVs are related to known OFC genes or pathways, but there are still many DNVs whose relevance to OFC development is unknown. To explore novel gene functions and their cellular expression profiles, we focused on DNVs in genes that were not listed in OMIM. We collected 960 DNVs in 853 genes from published studies and curated these genes, based on the DNVs’ deleteriousness, into 230 and 23 genes related to cleft lip with or without palate (CL/P) and cleft palate only (CPO), respectively. For comparison, we curated 178 CL/P and 277 CPO genes from OMIM. In CL/P, the pathways enriched in DNV and OMIM genes were significantly overlapped (p = 0.002). Single-cell RNA sequencing (scRNA-seq) analysis of mouse lip development revealed that both gene sets had abundant expression in the ectoderm (DNV genes: adjusted p = 0.032, OMIM genes: adjusted p < 0.0002), while only DNV genes were enriched in the endothelium (adjusted p = 0.032). Although we did not achieve significant findings using CPO gene sets, which was mainly due to the limited number of DNV genes, scRNA-seq analysis implicated various expression patterns among DNV and OMIM genes. Our results suggest combinatory pathway and scRNA-seq data analyses are helpful for contextualizing genes in OFC development.

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.

Sjögren’s disease (SjD) is a systemic autoimmune disorder characterized by dry eyes and mouth caused by chronic inflammation and is often accompanied by various extra-glandular manifestations, including fatigue and diffuse pain. Although the pathogenesis of the disease remains elusive, several factors (e.g. environmental, genetic and hormonal factors, abnormal metabolic status) are associated with this condition. Accumulating evidence suggests a potential role of cholesterol metabolism in immune and non-immune modulation in various diseases. In this review, we summarize the current findings on the associations between cholesterol metabolism and SjD.

Frontonasal malformations are caused by a failure in the growth of the frontonasal prominence during development. Although genetic studies have identified genes that are crucial for frontonasal development, it remains largely unknown how these genes are regulated during this process. Here, we show that microRNAs, which are short non-coding RNAs capable of targeting their target mRNAs for degradation or silencing their expression, play a crucial role in the regulation of genes related to frontonasal development in mice. Using the Mouse Genome Informatics (MGI) database, we curated a total of 25 mouse genes related to frontonasal malformations, including frontonasal hypoplasia, frontonasal dysplasia, and hypotelorism. MicroRNAs regulating the expression of these genes were predicted through bioinformatic analysis. We then experimentally evaluated the top three candidate miRNAs (miR-338-5p, miR-653-5p, and miR-374c-5p) for their effect on cell proliferation and target gene regulation in O9-1 cells, a neural crest cell line. Overexpression of these miRNAs significantly inhibited cell proliferation, and the genes related to frontonasal malformations (Alx1, Lrp2, and Sirt1 for miR-338-5p; Alx1, Cdc42, Sirt1, and Zic2 for miR-374c-5p; and Fgfr2, Pgap1, Rdh10, Sirt1, and Zic2 for miR-653-5p) were directly regulated by these miRNAs in a dose-dependent manner. Taken together, our results highlight miR-338-5p, miR-653-5p, and miR-374c-5p as pathogenic miRNAs related to the development of frontonasal malformations.

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.

Supplementary Figure 1 from Cathepsin E Prevents Tumor Growth and Metastasis by Catalyzing the Proteolytic Release of Soluble TRAIL from Tumor Cell Surface

Craniofacial deformities are one of the most common congenital birth defects [1; 2]. Several important 13 milestones in developing concepts, technologies, and methodologies in craniofacial biology have been 14achieved using animal models and transgenic technology. In fact, an increasing number of genetic and 15 epigenetic studies show temporospatial mechanisms underlying craniofacial development by using 16animal models such as genetically-engineered animal models and chemical-induced disease models

Computational learning especially, deep learning and machine learning have a huge impact. This current research topic gathered two fundamental concepts, deep learning and machine learning approaches applied to array-based biomedical data such as next generation sequencing (NGS) and medical imaging data.

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/β-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/β-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/β-catenin signaling pathways.

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.

Craniofacial structures change dynamically in morphology during development through the coordinated regulation of various cellular molecules. However, it remains unclear how these complex mechanisms are regulated in a spatiotemporal manner. Here we applied natural cubic splines to model gene and microRNA (miRNA) expression from embryonic day (E) 10.5 to E14.5 in the proximal and distal regions of the maxillary processes to identify spatiotemporal patterns of gene and miRNA expression, followed by constructing corresponding regulatory networks. Three major groups of differentially expressed genes (DEGs) were identified, including 3,927 temporal, 314 spatial, and 494 spatiotemporal DEGs. Unsupervised clustering further resolved these spatiotemporal DEGs into 8 clusters with distinct expression patterns. Interestingly, we found 2 clusters of differentially expressed miRNAs: 1 had 80 miRNAs monotonically decreasing and the other had 97 increasing across developmental stages. To evaluate the phenotypic relevance of these DEGs during craniofacial development, we integrated data from the CleftGeneDB database and constructed the regulatory networks of genes related to orofacial clefts. Our analysis revealed 2 hub miRNAs, mmu-miR-325-3p and mmu-miR-384-5p, that repressed cleft-related genes Adamts3, Runx2, Fgfr2, Acvr1, and Edn2, while their expression increased over time. On the contrary, 2 hub miRNAs, mmu-miR-218-5p and mmu-miR-338-5p, repressed cleft-related genes Pbx2, Ermp1, Snai1, Tbx2, and Bmi1, while their expression decreased over time. Our experiments indicated that these miRNA mimics significantly inhibited cell proliferation in mouse embryonic palatal mesenchymal (MEPM) cells and O9-1 cells through the regulation of genes associated with cleft palate and validated the role of our regulatory networks in orofacial clefts. To facilitate interactive exploration of these data, we developed a user-friendly web tool to visualize the gene and miRNA expression patterns across developmental stages, as well as the regulatory networks (https://fyan.shinyapps.io/facebase_shiny/). Taken together, our results provide a valuable resource that serves as a reference map for future research in craniofacial development.

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.

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 miR340-5p. 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.