Search Results (38)

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. Full article

Jumping translocations (JTs) can lead to partial trisomies. A breakpoint within the gene known as Jumping Translocation Breakpoint (JTB) has previously been associated with JTs involving the long arm of human chromosome 1 (1q). These 1q+ amplifications are frequently observed in cancer. JTB was initially mapped to the Epidermal Differentiation Complex (EDC) at 1q21, and earlier studies primarily focused on its role in malignant or adult tissues. Using updated genomic data, we refined the mapping of JTB. We employed RNA in situ hybridization (RISH) to visualize Jtb expression with organ, tissue, and cell-level resolution. We demonstrate that human JTB and murine Jtb are located outside the EDC. In midgestational wild-type mouse embryos, Jtb is expressed in multiple tissues, including the developing heart and vertebral column, and shows partial overlap with the expression of early markers of the neural crest cell lineage. Our findings suggest that the oncogenic potential associated with human JTB translocations is likely unrelated to its previously assumed location within the EDC. Full article

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. Full article

Although the roles of proteoglycans (PGs) have been well documented in the development and homeostasis of the temporomandibular joint (TMJ), how the glycosaminoglycan (GAG) chains of PGs contribute to TMJ chondrogenesis and osteogenesis still requires explication. In this study, we found that FAM20B, a hexokinase essential for attaching GAG chains to the core proteins of PGs, was robustly activated in the condylar mesenchyme during TMJ development. The inactivation of Fam20b in craniofacial neural crest cells (CNCCs) dramatically reduced the synthesis and accumulation of GAG chains rather than core proteins in the condylar cartilage, which resulted in a hypoplastic condylar cartilage by severely promoting chondrocyte hypertrophy and perichondral ossification. In the condyles of Wnt1-Cre;Fam20b^f/f mouse embryos, enlarged Ihh- and COL10-expressing domains indicated premature hypertrophy resulting from an attenuated IHH-PTHRP negative feedback in condylar chondrocytes, while increased osteogenic markers, canonical Wnt activity, and type-H angiogenesis verified the enhanced osteogenesis in the perichondrium. Further ex vivo investigations revealed that the loss of Fam20b decreased the domain area but increased the activity of HH signaling in the embryonic condylar mesenchyme. Moreover, the abrogation of GAG chains in heparan sulfate and chondroitin sulfate proteoglycans led to a rapid up- and then downregulation of HH signaling in condylar chondrocytes, implicating a “slow-release” manner of growth factors controlled by GAG chains. Overall, this study revealed a comprehensive role of the FAM20B-catalyzed GAG chain synthesis in the chondrogenic and osteogenic differentiation of the embryonic TMJ condyle. Full article

Adenosine deaminase acting on RNA 1 (ADAR1) is the principal enzyme for the adenosine-to-inosine RNA editing that prevents the aberrant activation of cytosolic nucleic acid sensors by endogenous double stranded RNAs and the activation of interferon-stimulated genes. In mice, the conditional neural crest deletion of Adar1 reduces the survival of melanocytes and alters the differentiation of Schwann cells that fail to myelinate nerve fibers in the peripheral nervous system. These myelination defects are partially rescued upon the concomitant removal of the Mda5 antiviral dsRNA sensor in vitro, suggesting implication of the Mda5/Mavs pathway and downstream effectors in the genesis of Adar1 mutant phenotypes. By analyzing RNA-Seq data from the sciatic nerves of mouse pups after conditional neural crest deletion of Adar1 (Adar1cKO), we here identified the transcription factors deregulated in Adar1cKO mutants compared to the controls. Through Adar1;Mavs and Adar1cKO;Egr1 double-mutant mouse rescue analyses, we then highlighted that the aberrant activation of the Mavs adapter protein and overexpression of the early growth response 1 (EGR1) transcription factor contribute to the Adar1 deletion associated defects in Schwann cell development in vivo. In silico and in vitro gene regulation studies additionally suggested that EGR1 might mediate this inhibitory effect through the aberrant regulation of EGR2-regulated myelin genes. We thus demonstrate the role of the Mda5/Mavs pathway, but also that of the Schwann cell transcription factors in Adar1-associated peripheral myelination defects. Full article

Background/Objectives: The microphthalmia-associated transcription factor (Mitf) has been found to play an important role in eye development, structure, and function. The Mitf gene is responsible for controlling cellular processes in a range of cell types, contributing to multiple eye development processes. In this review, we survey what is now known about the impact of Mitf on eye structure and function in retinal disorders. Several mutations in the human and mouse Mitf gene are now known, and the effects of these on eye phenotype are addressed. We discuss the importance of Mitf in regulating ion transport across the retinal pigment epithelium (RPE) and the vasculature of the eye. Methods: The literature was searched using the PubMed, Scopus, and Google Scholar databases. Fundus and Optical Coherence Tomography (OCT) images from mice were obtained with a Micron IV rodent imaging system. Results: Defects in neural-crest-derived melanocytes resulting from any Mitf mutations lead to hypopigmentation in the eye, coat, and inner functioning of the animals. While many Mitf mutations target RPE cells in the eye, fewer impact osteoclasts at the same time. Some of the mutations in mice lead to microphthalmia, and ultimately vision loss, while other mice show a normal eye size; however, the latter, in some cases, show hypopigmentation in the fundus and the choroid is depigmented and thickened, and in rare cases Mitf mutations lead to progressive retinal degeneration. Conclusions: The Mitf gene has an impact on the structure and function of the retina and its vasculature, the RPE, and the choroid in the adult eye. Full article

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. Full article

Cherubism is nowadays classified as an autoimmune disease and was first described in 1933. Although suspected at that time to be the result of defective tooth development, it was primarily classified as a bone disease caused by a mutation in the SH3BP2 gene. Despite a knock-in mouse model, phenotypic signs in the jaw area were not reproducible in this model. The features of classical cherubism can be attributed to a disturbed formation of the dental placode of the second molar. Since 2019, it has become clear that inhibition of the WNT pathway leads to the accumulation of SH3BP2 via tankyrase inhibition. As the dental placode is triggered via WNT (in epithelia) and MSX1 (in mesenchyme), aplasia of the second and third molars occurs due to a block in the WNT pathway. The mesenchymal part, which occurs prior to the body plan regulation of the WNT/MSX1 pathway, remains unaffected and provides the substrate for the giant cell granuloma. Considering macrophage polarization and the role of the extracellular matrix in general, cherubism is situated in the field of tension between autoimmune diseases and cancer. In this sense, we see the cause of cherubism in a WNT-related dysregulation, which can be proven postnatally in the neural crest-related tooth development of the replacement tooth ridge, both genotypically and phenotypically. Full article

Dental pulp pericytes are reported to have the capacity to generate odontoblasts and express multiple cytokines and chemokines that regulate the local immune microenvironment, thus participating in the repair of dental pulp injury in vivo. However, it has not yet been reported whether the transplantation of exogenous pericytes can effectively treat pulpitis, and the underlying molecular mechanism remains unknown. In this study, using a lineage-tracing mouse model, we showed that most dental pulp pericytes are derived from cranial neural crest. Then, we demonstrated that the ablation of pericytes could induce a pulpitis-like phenotype in uninfected dental pulp in mice, and we showed that the significant loss of pericytes occurs during pupal inflammation, implying that the transplantation of pericytes may help to restore dental pulp homeostasis during pulpitis. Subsequently, we successfully generated pericytes with immunomodulatory activity from human pluripotent stem cells through the intermediate stage of the cranial neural crest with a high level of efficiency. Most strikingly, for the first time we showed that, compared with the untreated pulpitis group, the transplantation of hPSC-derived pericytes could substantially inhibit vascular permeability (the extravascular deposition of fibrinogen, ** p < 0.01), alleviate pulpal inflammation (TCR⁺ cell infiltration, * p < 0.05), and promote the regeneration of dentin (** p < 0.01) in the mouse model of pulpitis. In addition, we discovered that the knockdown of latent transforming growth factor beta binding protein 1 (LTBP1) remarkably suppressed the immunoregulation ability of pericytes in vitro and compromised their in vivo regenerative potential in pulpitis. These results indicate that the transplantation of pericytes could efficiently rescue the aberrant phenotype of pulpal inflammation, which may be partially due to LTBP1-mediated T cell suppression. Full article

Insights into the role of microRNAs (miRNAs) in disease pathogenesis have made them attractive therapeutic targets, and numerous miRNAs have been functionally linked to Hirschsprung disease (HSCR), a life-threatening genetic disorder due to defective migration, proliferation, and colonization of enteric neural crest cells (ENCCs) in the gut. Recent studies have demonstrated that miR-424 strongly inhibits migration in a variety of cell types and its potential target RICTOR is essential for neural crest cell development. We therefore sought to interrogate how miR-424 and RICTOR contribute to the pathogenesis of HSCR. We utilized HSCR cases and human neural cells to evaluate the miR-424-mediated regulation of RICTOR and the downstream AKT phosphorylation. We further developed an ex vivo model to assess the effects of miR-424 on ENCC migration and proliferation. Then, single-cell atlases of gene expression in both human and mouse fetal intestines were used to determine the characteristics of RICTOR and AKT expression in the developing gut. Our findings demonstrate that miR-424 levels are markedly increased in the colonic tissues of patients with HSCR and that it regulates human neural cell migration by directly targeting RICTOR. Up-regulation of miR-424 leads to decreased AKT phosphorylation levels in a RICTOR-dependent manner, and this, in turn, impairs ENCC proliferation and migration in the developing gut. Interestingly, we further identified prominent RICTOR and AKT expressions in the enteric neurons and other types of enteric neural cells in human and mouse fetal intestines. Our present study reveals the role of the miR-424/RICTOR axis in HSCR pathogenesis and indicates that miR-424 is a promising candidate for the development of targeted therapies against HSCR. Full article

Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology. Full article

Hirschsprung disease (HSCR) is a complex congenital disorder caused by defects in the development of the enteric nervous system (ENS). It is attributed to failures of the enteric neural crest stem cells (ENCCs) to proliferate, differentiate and/or migrate, leading to the absence of enteric neurons in the distal colon, resulting in colonic motility dysfunction. Due to the oligogenic nature of the disease, some HSCR conditions could not be phenocopied in animal models. Building the patient-based disease model using human induced pluripotent stem cells (hPSC) has opened up a new opportunity to untangle the unknowns of the disease. The expanding armamentarium of hPSC-based therapies provides needed new tools for developing cell-replacement therapy for HSCR. Here we summarize the recent studies of hPSC-based models of ENS in 2-D and 3-D culture systems. These studies have highlighted how hPSC-based models complement the population-based genetic screens and bioinformatic approaches for the discovery of new HSCR susceptibility genes and provide a human model for the close-to-physiological functional studies. We will also discuss the potential applications of these hPSC-based models in translational medicines and their advantages and limitations. The use of these hPSC-based models for drug discovery or cell replacement therapy likely leads to new treatment strategies for HSCR in the future. Further improvements in incorporating hPSC-based models with the human-mouse chimera model and organ-on-a-chip system for establishing a better disease model of HSCR and for drug discovery will further propel us to success in the development of an efficacious treatment for HSCR. Full article

Persistent truncus arteriosus (PTA) is an uncommon and complex congenital cardiac malformation accounting for about 1.2% of all congenital heart diseases (CHDs), which is caused by a deficiency in the embryonic heart outflow tract’s (OFT) septation and remodeling. PDGFRα and PDGFRβ double knockout (DKO) in cardiac neural crest cells (CNCCs) has been reported to cause PTA, but the underlying mechanisms remain unclear. Here, we constructed a PTA mouse model with PDGFRα and PDGFRβ double knockout in Pax3⁺ CNCCs and described the condensation failure into OFT septum of CNCC-derived cells due to disturbance of cell polarity in the DKO group. In addition, we further explored the mechanism with single-cell RNA sequencing. We found that two main cell differentiation trajectories into vascular smooth muscle cells (VSMCs) from cardiomyocytes (CMs) and mesenchymal cells (MSs), respectively, were interrupted in the DKO group. The process of CM differentiation into VSMC stagnated in a transitional CM I-like state, which contributed to the failure of OFT remodeling and muscular septum formation. On the other hand, a Penk⁺ transitional MS II cluster closely related to cell condensation into the OFT septum disappeared, which led to the OFT’s septation absence directly. In conclusion, the disturbance of CNCC-derived cells caused by PDGFRα and PDGFRβ knockout can lead to the OFT septation disorder and the occurrence of PTA. Full article

Branchiomeric skeletal muscles are a subset of head muscles originating from skeletal muscle progenitor cells in the mesodermal core of pharyngeal arches. These muscles are involved in facial expression, mastication, and function of the larynx and pharynx. Branchiomeric muscles have been the focus of many studies over the years due to their distinct developmental programs and common origin with the heart muscle. A prerequisite for investigating these muscles’ properties and therapeutic potential is understanding their genetic program and differentiation. In contrast to our understanding of how branchiomeric muscles are formed, less is known about their differentiation. This review focuses on the differentiation of branchiomeric muscles in mouse embryos. Furthermore, the relationship between branchiomeric muscle progenitor and neural crest cells in the pharyngeal arches of chicken embryos is also discussed. Additionally, we summarize recent studies into the genetic networks that distinguish between first arch-derived muscles and other pharyngeal arch muscles. Full article

Carbamazepine (CBZ, Tegretol^®) is an anticonvulsant used in the treatment of epilepsy and neuropathic pain; however, several unwanted effects of this drug have been noticed. Therefore, the regulatory actions of CBZ on ionic currents in electrically excitable cells need to be reappraised, although its efficacy in suppressing voltage-gated Na⁺ current (I_Na) has been disclosed. This study was undertaken to explore the modifications produced by CBZ on ionic currents (e.g., I_Na and erg-mediated K⁺ current [I_K(erg)]) measured from Neuro-2a (N2a) cells. In these cells, we found that this drug differentially suppressed the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of I_Na in a concentration-dependent manner with effective IC₅₀ of 56 and 18 μM, respectively. The overall current–voltage relationship of I_Na(T) with or without the addition of CBZ remained unchanged; however, the strength (i.e., ∆area) in the window component of I_Na (I_Na(W)) evoked by the short ascending ramp pulse (V_ramp) was overly lessened in the CBZ presence. Tefluthrin (Tef), a synthetic pyrethroid, known to stimulate I_Na, augmented the strength of the voltage-dependent hysteresis (Hys_(V)) of persistent I_Na (I_Na(P)) in response to the isosceles-triangular V_ramp; moreover, further application of CBZ attenuated Tef-mediated accentuation of I_Na(P)’s Hys_(V). With a two-step voltage protocol, the recovery of I_Na(T) inactivation seen in Neuro-2a cells became progressively slowed by adding CBZ; however, the cumulative inhibition of I_Na(T) evoked by pulse train stimulation was enhanced during exposure to this drug. Neuro-2a-cell exposure to CBZ (100 μM), the magnitude of erg-mediated K⁺ current measured throughout the entire voltage-clamp steps applied was mildly inhibited. The docking results regarding the interaction of CBZ and voltage-gate Na⁺ (Na_V) channel predicted the ability of CBZ to bind to some amino-acid residues in Na_V due to the existence of a hydrogen bond or hydrophobic contact. It is conceivable from the current investigations that the I_Na (I_Na(T), I_Na(L), I_Na(W), and I_Na(P)) residing in Neuro-2a cells are susceptible to being suppressed by CBZ, and that its block on I_Na(L) is larger than that on I_Na(T). Collectively, the magnitude and gating of Na_V channels produced by the CBZ presence might have an impact on its anticonvulsant and analgesic effects occurring in vivo. Full article