ABSTRACT
Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While granule cell cilia are essential during early developmental stages, they become infrequent upon maturation. Here, we provide nanoscopic resolution of cilia in situ using large-scale electron microscopy volumes and immunostaining of mouse cerebella. In many granule cells, we found intracellular cilia, concealed from the external environment. Cilia were disassembled in differentiating granule cell neurons-in a process we call cilia deconstruction-distinct from premitotic cilia resorption in proliferating progenitors. In differentiating granule cells, cilia deconstruction involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Unlike ciliated neurons in other brain regions, our results show the deconstruction of concealed cilia in differentiating granule cells, which might prevent mitogenic hedgehog responsiveness. Ciliary deconstruction could be paradigmatic of cilia removal during differentiation in other tissues.
Subject(s)
Cell Differentiation , Cerebellum , Cilia , Hedgehog Proteins , Neurons , Cilia/metabolism , Cilia/ultrastructure , Animals , Neurons/metabolism , Neurons/cytology , Neurons/ultrastructure , Mice , Cerebellum/metabolism , Cerebellum/cytology , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Neurogenesis , Centrioles/metabolism , Centrioles/ultrastructure , Mice, Inbred C57BLABSTRACT
The Basal Cell Carcinoma (BCC) is a sort of unique tumour due to its combined peculiar histological features and clinical behaviour, such as the constant binary involvement of the epithelium and the stroma, the virtual absence of metastases and the predilection of specific anatomical sites for both onset and spread. A potential correlation between the onset of BCC and a dysembryogenetic process has long been hypothesised. A selective investigation of PubMed-indexed publications supporting this theory retrieved 64 selected articles published between 1901 and 2024. From our analysis of the literature review, five main research domains on the dysembryogenetic pathogenesis of BCC were identified: (1) The correlation between the topographic distribution of BCC and the macroscopic embryology, (2) the correlation between BCC and the microscopic embryology, (3) the genetic BCC, (4) the correlation between BCC and the hair follicle and (5) the correlation between BCC and the molecular embryology with a specific focus on the Hedgehog signalling pathway. A large amount of data from microscopic and molecular research consistently supports the hypothesis of a dysembryogenetic pathogenesis of BCC. Such evidence is promoting advances in the clinical management of this disease, with innovative targeted molecular therapies on an immune modulating basis being developed.
Subject(s)
Carcinoma, Basal Cell , Hedgehog Proteins , Skin Neoplasms , Carcinoma, Basal Cell/pathology , Carcinoma, Basal Cell/etiology , Carcinoma, Basal Cell/genetics , Humans , Skin Neoplasms/pathology , Skin Neoplasms/genetics , Skin Neoplasms/etiology , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Signal Transduction , Hair Follicle/pathology , Hair Follicle/embryology , Hair Follicle/metabolismABSTRACT
Sonic hedgehog subgroup of medulloblastoma (SHH-MB) is characterized by aberrant activation of the SHH signaling pathway. An inhibition of the positive SHH regulator Smoothened (SMO) has demonstrated promising clinical efficacy. Yet, primary and acquired resistance to SMO inhibitors limit their efficacy. An understanding of underlying molecular mechanisms of resistance to therapy is warranted to bridge this unmet need. Here, we make use of genome-wide CRISPR-Cas9 knockout screens in murine SMB21 and human DAOY cells, in order to unravel genetic dependencies and drug-related genetic interactors that could serve as alternative therapeutic targets for SHH-MB. Our screens reinforce SMB21 cells as a faithful model system for SHH-MB, as opposed to DAOY cells, and identify members of the epigenetic machinery including DNA methyltransferase 1 (DNMT1) as druggable targets in SHH-dependent tumors. We show that Dnmt1 plays a crucial role in normal murine cerebellar development and is required for SHH-MB growth in vivo. Additionally, DNMT1 pharmacological inhibition alone and in combination with SMO inhibition effectively inhibits tumor growth in murine and human SHH-MB cell models and prolongs survival of SHH-MB mouse models by inhibiting SHH signaling output downstream of SMO. In conclusion, our data highlight the potential of inhibiting epigenetic regulators as a novel therapeutic avenue in SMO-inhibitor sensitive as well as resistant SHH-MBs.
Subject(s)
CRISPR-Cas Systems , Cerebellar Neoplasms , DNA (Cytosine-5-)-Methyltransferase 1 , Hedgehog Proteins , Medulloblastoma , Medulloblastoma/genetics , Medulloblastoma/metabolism , Medulloblastoma/pathology , Animals , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/metabolism , Cerebellar Neoplasms/pathology , Humans , Mice , Cell Line, Tumor , Smoothened Receptor/genetics , Smoothened Receptor/metabolism , Gene Knockout Techniques/methodsABSTRACT
Liver cancers, including hepatocellular carcinoma (HCC), are the sixth most common cancer and the third leading cause of cancer-related death worldwide, representing a global public health problem. This study evaluated nine patients with HCC. Six of the cases involved hepatic explants, and three involved hepatic segmentectomy for tumor resection. Eight out of nine tumors were HCC, with one being a combined hepatocellular-cholangiocarcinoma tumor. Conventional markers of hepatocellular differentiation (Hep Par-1, arginase, pCEA, and glutamine synthetase) were positive in all patients, while markers of hepatic precursor cells (CK19, CK7, EpCAM, and CD56) were negative in most patients, and when positive, they were detected in small, isolated foci. Based on in silico analysis of HCC tumors from The Cancer Genome Atlas database, we found that Hedgehog (HH) pathway components (GLI1, GLI2, GLI3 and GAS1) have high connectivity values (module membership > 0.7) and are strongly correlated with each other and with other genes in biologically relevant modules for HCC. We further validated this finding by analyzing the gene expression of HH components (PTCH1, GLI1, GLI2 and GLI3) in our samples through qPCR, as well as by immunohistochemical analysis. Additionally, we conducted a chemosensitivity analysis using primary HCC cultures treated with a panel of 18 drugs that affect the HH pathway and/or HCC. Most HCC samples were sensitive to sunitinib. Our results offer a comprehensive view of the molecular landscape of HCC, highlighting the significance of the HH pathway and providing insight into focused treatments for HCC.
Subject(s)
Carcinoma, Hepatocellular , Hedgehog Proteins , Liver Neoplasms , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Female , Male , Middle Aged , Aged , Gene Expression Regulation, Neoplastic , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/genetics , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein GLI1/genetics , Signal Transduction , Sunitinib/pharmacology , Sunitinib/therapeutic use , Adult , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/geneticsABSTRACT
In years of research on classical pathways, the composition, information transmission mechanism, crosstalk with other pathways, and physiological and pathological effects of hedgehog (HH) pathway have been gradually clarified. HH also plays a critical role in tumor formation and development. According to the update of interpretation of tumor phenotypes, the latest relevant studies have been sorted out, to explore the specific mechanism of HH pathway in regulating different tumor phenotypes through gene mutation and signal regulation. The drugs and natural ingredients involved in regulating HH pathway were also reviewed; five approved drugs and drugs under research exert efficacy by blocking HH pathway, and at least 22 natural components have potential to treat tumors by HH pathway. Nevertheless, there is a deficiency of existing studies. The present review confirmed the great potential of HH pathway in future cancer treatment with factual basis.
Subject(s)
Hedgehog Proteins , Neoplasms , Signal Transduction , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , Signal Transduction/drug effects , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/pharmacology , Animals , MutationABSTRACT
Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-creKI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-creKI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-creKI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-creKI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-creKI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-creKI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia.
Subject(s)
Fibroblast Growth Factor 8 , Incisor , Mesoderm , Molar , Animals , Fibroblast Growth Factor 8/genetics , Fibroblast Growth Factor 8/metabolism , Mice , Incisor/abnormalities , Incisor/metabolism , Mesoderm/metabolism , Mesoderm/pathology , Molar/abnormalities , Molar/metabolism , Anodontia/genetics , Anodontia/metabolism , Anodontia/pathology , Apoptosis , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Lymphoid Enhancer-Binding Factor 1/metabolism , Lymphoid Enhancer-Binding Factor 1/genetics , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Signal Transduction , Gene Expression Regulation, Developmental , Odontogenesis/genetics , Mice, TransgenicABSTRACT
The functional maturation of the pituitary gland requires adequate cell differentiation and vascular network formation. Although spatiotemporal signaling and transcription factors are known to govern pituitary development, the involvement of primary cilia, nonmoving hair-like organelles, remains unclear. In this study, we uncovered the contribution of primary cilia to cell-type determination and vascular network formation during pituitary development. Homozygous knockout mice lacking a ciliary kinase, Dyrk2-/-, exhibit abnormalities in ciliary structure and pituitary hypoplasia, accompanied by varying degrees of failure in differentiation among all types of hormone-producing cells in the anterior lobe. Aberrations in cell differentiation in Dyrk2-/- mice arise from a decrease in the expression of crucial transcription factors, Lhx4, Lhx3, and Prop1, resulting from the inactivity of Hedgehog (Hh) signaling during the early stages of development. Furthermore, the loss of Dyrk2 results in vascular system abnormalities during the middle to late stages of development. Mechanistically, transcriptome analyses revealed the downregulation of vitronectin-integrin αvß3-VEGFR2 signaling, essential for orchestrating vascular development. Collectively, our findings demonstrate that primary cilia play a pivotal role as critical regulators of cell survival, cell determination, and angiogenesis during pituitary gland development through the activation of Hh signaling. These findings expand our understanding of the potential link between pituitary dysfunction in human disorders and ciliopathies.
Subject(s)
Cell Differentiation , Cilia , Neovascularization, Physiologic , Pituitary Gland , Animals , Mice , Angiogenesis , Cilia/metabolism , Cilia/physiology , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Mice, Knockout , Neovascularization, Physiologic/genetics , Neovascularization, Physiologic/physiology , Pituitary Gland/metabolism , Signal Transduction , Transcription Factors/metabolism , Transcription Factors/genetics , Dyrk Kinases/geneticsABSTRACT
AIMS: Colon cancer poses a major threat to human health and a heavy burden on the national economy. As a member of the SOX transcription factor family, SRY-box transcription factor 21 (SOX21) is associated with various cancers, but its mechanism of action in colon cancer remains unclear. This study focused on the molecular mechanisms of transcription factor SOX21 in proliferation and metastasis of colon cancer cells. MAIN METHODS: We analyzed SOX21 expression level and its impact on survival in colon cancer patients by bioinformatics analysis. We used public databases for gene correlation, GSEA enrichment analysis. Cell function experiments (colony formation assay, wound healing assay, Transwell migration and invasion assay) were utilized to determine the impact of SOX21 silencing and over-expression on cell proliferation and metastasis. The luciferase reporter assay, CUT&RUN-qPCR assay and Methylation Specific PCR were used to explore SOX21-POU class 4 homeobox 2 (POU4F2) molecular interactions. The molecular mechanisms were verified by Quantitative real-time PCR and Western blot analysis. KEY FINDINGS: SOX21 is highly expressed and affects the overall survival of colon cancer patients. SOX21 can attenuates POU4F2 methylation state by binding with it. In addition, this interaction facilitate its transcriptional activation of Hedgehog pathway, mediates epithelial-mesenchymal transition (EMT), consequently promoting the proliferation and metastasis of colon cancer cells. SIGNIFICANCE: Our study reveals that SOX21 is an oncogenic molecule and suggests its regulatory role in colon carcinogenesis and progression, providing new insights into the treatment of this disease.
Subject(s)
Cell Proliferation , Colonic Neoplasms , Epithelial-Mesenchymal Transition , Hedgehog Proteins , Signal Transduction , Humans , Epithelial-Mesenchymal Transition/genetics , Colonic Neoplasms/pathology , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Neoplasm Metastasis , Cell Movement , SOXB2 Transcription Factors/metabolism , SOXB2 Transcription Factors/genetics , Octamer Transcription Factor-3/metabolism , Octamer Transcription Factor-3/geneticsABSTRACT
BACKGROUND: Trans-sutural distraction osteogenesis (TSDO) involves the application of distraction force to facial sutures to stimulate osteogenesis. Gli1+ cells in the cranial sutures play an important role in bone growth. However, whether Gli1+ cells in facial sutures differentiate into bone under distraction force is unknown. METHODS: 4-week-old Gli1ER/Td and C57BL/6 mice were used to establish a TSDO model to explore osteogenesis of zygomaticomaxillary sutures. A Gli1+ cell lineage tracing model was used to observe the distribution of Gli1+ cells and explore the role of Gli1+ cells in facial bone remodeling. RESULTS: Distraction force promoted bone remodeling during TSDO. Fluorescence and two-photon scanning images revealed the distribution of Gli1+ cells. Under distraction force, Gli1-lineage cells proliferated significantly and co-localized with Runx2+ cells. Hedgehog signaling was upregulated in Gli1+ cells. Inhibition of Hedgehog signaling suppresses the proliferation and osteogenesis of Gli1+ cells induced by distraction force. Subsequently, the stem cell characteristics of Gli1+ cells were identified. Cell-stretching experiments verified that mechanical force promoted the osteogenic differentiation of Gli1+ cells through Hh signaling. Furthermore, immunofluorescence staining and RT-qPCR experiments demonstrated that the primary cilia in Gli1+ cells exhibit Hedgehog-independent mechanosensitivity, which was required for the osteogenic differentiation induced by mechanical force. CONCLUSIONS: Our study indicates that the primary cilia of Gli1+ cells sense mechanical stimuli, mediate Hedgehog signaling activation, and promote the osteogenic differentiation of Gli1+ cells in zygomaticomaxillary sutures.
Subject(s)
Cell Differentiation , Cilia , Cranial Sutures , Hedgehog Proteins , Osteogenesis , Signal Transduction , Zinc Finger Protein GLI1 , Animals , Mice , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein GLI1/genetics , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Osteogenesis/physiology , Cilia/metabolism , Cranial Sutures/metabolism , Mice, Inbred C57BL , Osteogenesis, Distraction/methods , Cell ProliferationABSTRACT
Purpose: Microphthalmia is a rare developmental eye disease that affects 1 in 7000 births. Currently, there is no cure for this condition. This study aimed to construct a stable mouse model of microphthalmia, thus providing a new tool for the study of the etiology of microphthalmia. Methods: The Hedgehog signaling pathway plays a crucial role in eye development. One of the key mechanisms of the Sonic Hedgehog signaling is the strong transcriptional activation ability of GLI3, a major mediator of this pathway. This study used CRISPR/Cas9 system to construct a novel TgGli3Ki/Ki lens-specific over-expression mouse line. To identify the ocular characteristics of this line, quantitative PCR, Western blot, hematoxylin and eosin staining, immunofluorescent staining, and RNA-seq were performed on the ocular tissues of this line and normal mice. Results: The TgGli3Ki/Ki lens-specific over-expression mouse model exhibits the ocular phenotype of microphthalmia. In the TgGli3Ki/Ki mouse, Gli3 is over-expressed in the lens, and the size of the eyeball and lens is significantly smaller than the normal one. RNA-seq analysis using the lens and the retina samples from TgGli3Ki/Ki and normal mice indicates that the phototransduction pathway is ectopically activated in the lens. Immunofluorescent staining of the lens samples confirmed this activation. Conclusions: The TgGli3Ki/Ki mouse model consistently manifests the stereotypical microphthalmia phenotype across generations, making it an excellent tool for studying this severe eye disease. Translational Relevance: This study developed a novel animal model to facilitate clinical research on microphthalmia.
Subject(s)
Disease Models, Animal , Microphthalmos , Zinc Finger Protein Gli3 , Animals , Microphthalmos/genetics , Microphthalmos/pathology , Microphthalmos/metabolism , Mice , Zinc Finger Protein Gli3/genetics , Zinc Finger Protein Gli3/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Lens, Crystalline/metabolism , Lens, Crystalline/pathology , Signal Transduction , CRISPR-Cas Systems , Mice, Inbred C57BL , Nerve Tissue ProteinsABSTRACT
The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.
Subject(s)
Chondrocytes , Forkhead Transcription Factors , Growth Plate , Hypertrophy , Osteogenesis , Animals , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Chondrocytes/metabolism , Chondrocytes/cytology , Mice , Growth Plate/metabolism , Growth Plate/pathology , Growth Plate/embryology , Osteogenesis/genetics , Extremities/embryology , Extremities/pathology , Chondrogenesis/genetics , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Gene Expression Regulation, Developmental , Cell Differentiation , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Cartilage/metabolism , Cartilage/pathology , Cartilage/embryologyABSTRACT
Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.
Subject(s)
Dyrk Kinases , Hedgehog Proteins , Protein Serine-Threonine Kinases , Protein-Tyrosine Kinases , Signal Transduction , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3 , Animals , Zinc Finger Protein Gli3/metabolism , Zinc Finger Protein Gli3/genetics , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/genetics , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Mice , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Humans , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/genetics , Cell Proliferation , Cilia/metabolism , Smoothened Receptor/metabolism , Smoothened Receptor/genetics , Nuclear Proteins , Repressor ProteinsABSTRACT
Congenital lung malformations are fatal at birth in their severe forms. Prevention and early intervention of these birth defects require a comprehensive understanding of the molecular mechanisms of lung development. We find that the loss of inturned (Intu), a cilia and planar polarity effector gene, severely disrupts growth and branching morphogenesis of the mouse embryonic lungs. Consistent with our previous results indicating an important role for Intu in ciliogenesis and hedgehog (Hh) signaling, we find greatly reduced number of primary cilia in both the epithelial and mesenchymal tissues of the lungs. We also find significantly reduced expression of Gli1 and Ptch1, direct targets of Hh signaling, suggesting disruption of cilia-dependent Hh signaling in Intu mutant lungs. An agonist of the Hh pathway activator, smoothened, increases Hh target gene expression and tubulogenesis in explanted wild type, but not Intu mutant, lungs, suggesting impaired Hh signaling response underlying lung morphogenetic defects in Intu mutants. Furthermore, removing both Gli2 and Intu completely abolishes branching morphogenesis of the lung, strongly supporting a mechanism by which Intu regulates lung growth and patterning through cilia-dependent Hh signaling. Moreover, a transcriptomics analysis identifies around 200 differentially expressed genes (DEGs) in Intu mutant lungs, including known Hh target genes Gli1, Ptch1/2 and Hhip. Genes involved in muscle differentiation and function are highly enriched among the DEGs, consistent with an important role of Hh signaling in airway smooth muscle differentiation. In addition, we find that the difference in gene expression between the left and right lungs diminishes in Intu mutants, suggesting an important role of Intu in asymmetrical growth and patterning of the mouse lungs.
Subject(s)
Cilia , Gene Expression Regulation, Developmental , Hedgehog Proteins , Lung , Signal Transduction , Animals , Mice , Body Patterning/genetics , Cilia/metabolism , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Lung/embryology , Lung/metabolism , Morphogenesis/genetics , Patched-1 Receptor/metabolism , Patched-1 Receptor/genetics , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/geneticsABSTRACT
OBJECTIVE: Sarcopenia manifests as a chronic, low-level inflammation along with multiple inflammatory cells infiltration. Interleukin (IL)-25 can regulate the function of macrophages. However, the specific role and mechanisms through which IL-25 functions in sarcopenia are still not fully understood and require further investigation. METHODS: Aged mice were utilized as sarcopenia models and examined the expression of inflammatory factors. To investigate the effects of IL-25 on sarcopenia, the model mice received IL-25 treatment and underwent in vivo adoptive transfer of IL-25-induced macrophages. Meanwhile, RAW264.7 cells, bone marrow-derived macrophages, satellite cells and C2C12 cells were used in vitro. Shh insufficiency was induced through intramuscular administration of SHH-shRNA adenoviruses. Then, various assays including scratch wound, cell counting kit-8 and Transwell assays, as well as histological staining and molecular biological methods, were conducted. RESULTS: Aged mice exhibited an accelerated decline in muscle strength and mass, along with an increased muscle lipid droplets and macrophage infiltration, and decreased IL-25 levels compared to the young group. IL-25 therapy and the transfer of IL-25-preconditioned macrophages could improve these conditions by promoting M2 macrophage polarization in vivo as well as in vitro. M2 macrophage conditioned medium enhanced satellite cell proliferation and migration, as well as the vitality, migration, and differentiation of C2C12 cells in vitro. Furthermore, IL-25 enhanced Shh expression in macrophages in vitro, and activated the Shh signaling pathway in muscle tissue of aged mice, which could be suppressed by either the inhibitor cyclopamine or Shh knockdown. Mechanistic studies showed that Shh insufficiency suppressed the activation of Akt/mTOR signaling pathway in muscle tissue of aged mice. CONCLUSION: IL-25 promotes the secretion of Shh by M2 macrophages and activates the Shh/Akt/mTOR signaling pathway, which improves symptoms and function in sarcopenia mice. This suggests that IL-25 has potential as a therapeutic agent for treating sarcopenia.
Subject(s)
Hedgehog Proteins , Macrophages , Mice, Inbred C57BL , Muscle, Skeletal , Regeneration , Sarcopenia , Signal Transduction , Animals , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Mice , Macrophages/immunology , Macrophages/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscle, Skeletal/drug effects , RAW 264.7 Cells , Male , Interleukin-17/metabolism , Disease Models, Animal , Humans , Cell Proliferation/drug effectsABSTRACT
The direction of left-right visceral asymmetry is conserved in vertebrates. Deviations of the standard asymmetric pattern are rare, and the underlying mechanisms are not understood. Here, we use the teleost Astyanax mexicanus, consisting of surface fish with normal left-oriented heart asymmetry and cavefish with high levels of reversed right-oriented heart asymmetry, to explore natural changes in asymmetry determination. We show that Sonic Hedgehog (Shh) signaling is increased at the posterior midline, Kupffer's vesicle (the teleost left-right organizer) is enlarged and contains longer cilia, and the number of dorsal forerunner cells is increased in cavefish. Furthermore, Shh increase in surface fish embryos induces asymmetric changes resembling the cavefish phenotype. Asymmetric expression of the Nodal antagonist Dand5 is equalized or reversed in cavefish, and Shh increase in surface fish mimics changes in cavefish dand5 asymmetry. Shh decrease reduces the level of right-oriented heart asymmetry in cavefish. Thus, naturally occurring modifications in cavefish heart asymmetry are controlled by the effects of Shh signaling on left-right organizer function.
Subject(s)
Body Patterning , Heart , Hedgehog Proteins , Signal Transduction , Animals , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Body Patterning/genetics , Heart/embryology , Characidae/embryology , Characidae/genetics , Gene Expression Regulation, Developmental , Cilia/metabolism , Embryo, Nonmammalian/metabolismABSTRACT
The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.
Subject(s)
Cell Adhesion , Cell Movement , Hedgehog Proteins , Myosin Type II , Signal Transduction , Animals , Mice , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Cell Adhesion/genetics , Myosin Type II/metabolism , Myosin Type II/genetics , Cell Movement/genetics , Epithelium/metabolism , Morphogenesis/genetics , Tooth/metabolism , Tooth/growth & development , Epithelial Cells/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/genetics , Gene Expression Regulation, DevelopmentalABSTRACT
BACKGROUND: Heterozygous Indian Hedgehog gene (IHH) variants are associated with brachydactyly type A1 (BDA1). However, in recent years, numerous variants have been identified in patients with short stature and more variable forms of brachydactyly. Many are located in the C-terminal domain of IHH (IHH-C), which lacks signaling activity but is critical for auto-cleavage and activation of the N-terminal (IHH-N) peptide. The absence of functional studies of IHH variants, particularly for those located in IHH-C, has led to these variants being classified as variants of uncertain significance (VUS). OBJECTIVE: To establish a simple functional assay to determine the pathogenicity of IHH VUS and confirm that variants in the C-terminal domain affect protein function. DESIGN/METHODS: In vitro studies were performed for 9 IHH heterozygous variants, to test their effect on secretion and IHH intracellular processing by western blot of cells expressing each variant. RESULTS: IHH secretion was significantly reduced in all mutants, regardless of the location. Similarly, intracellular levels of N-terminal and C-terminal IHH peptides were severely reduced in comparison with the control. Two variants present at a relatively high frequency in the general population also reduced secretion but to a lesser degree in the heterozygous state. CONCLUSIONS: These studies provide the first evidence that variants in the C-terminal domain affect the secretion capacity of IHH and thus, reduce availability of IHH ligand, resulting in short stature and mild skeletal defects. The secretion assay permits a relatively easy test to determine the pathogenicity of IHH variants. All studied variants affected secretion and interestingly, more frequent population variants appear to have a deleterious effect and thus contribute to height variation.
Subject(s)
Hedgehog Proteins , Humans , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Protein Domains/genetics , Brachydactyly/genetics , Dwarfism/genetics , Mutation , Animals , Genetic Variation/genetics , Body Height/genetics , HeterozygoteABSTRACT
The Hedgehog (HH) pathway regulates embryonic development of anterior tongue taste fungiform papilla (FP) and the posterior circumvallate (CVP) and foliate (FOP) taste papillae. HH signaling also mediates taste organ maintenance and regeneration in adults. However, there are knowledge gaps in HH pathway component expression during postnatal taste organ differentiation and maturation. Importantly, the HH transcriptional effectors GLI1, GLI2 and GLI3 have not been investigated in early postnatal stages; the HH receptors PTCH1, GAS1, CDON and HHIP, required to either drive HH pathway activation or antagonism, also remain unexplored. Using lacZ reporter mouse models, we mapped expression of the HH ligand SHH, HH receptors, and GLI transcription factors in FP, CVP and FOP in early and late postnatal and adult stages. In adults we also studied the soft palate, and the geniculate and trigeminal ganglia, which extend afferent fibers to the anterior tongue. Shh and Gas1 are the only components that were consistently expressed within taste buds of all three papillae and the soft palate. In the first postnatal week, we observed broad expression of HH signaling components in FP and adjacent, non-taste filiform (FILIF) papillae in epithelium or stroma and tongue muscles. Notably, we observed elimination of Gli1 in FILIF and Gas1 in muscles, and downregulation of Ptch1 in lingual epithelium and of Cdon, Gas1 and Hhip in stroma from late postnatal stages. Further, HH receptor expression patterns in CVP and FOP epithelium differed from anterior FP. Among all the components, only known positive regulators of HH signaling, SHH, Ptch1, Gli1 and Gli2, were expressed in the ganglia. Our studies emphasize differential regulation of HH signaling in distinct postnatal developmental periods and in anterior versus posterior taste organs, and lay the foundation for functional studies to understand the roles of numerous HH signaling components in postnatal tongue development.
Subject(s)
Hedgehog Proteins , Signal Transduction , Taste Buds , Tongue , Animals , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Tongue/metabolism , Tongue/growth & development , Mice , Taste Buds/metabolism , Taste Buds/growth & development , Gene Expression Regulation, Developmental , Homeostasis , Patched-1 Receptor/metabolism , Patched-1 Receptor/genetics , Zinc Finger Protein GLI1/metabolism , Zinc Finger Protein GLI1/genetics , Kruppel-Like Transcription Factors/metabolism , Kruppel-Like Transcription Factors/genetics , Zinc Finger Protein Gli2/metabolism , Zinc Finger Protein Gli2/genetics , Zinc Finger Protein Gli3/metabolism , Zinc Finger Protein Gli3/genetics , Nerve Tissue Proteins , Cell Cycle Proteins , GPI-Linked ProteinsABSTRACT
The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we show that Spinal Cord Organoids (SCOs) can be applied to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. We validate this system by measuring the effects of mutating the HH receptor PTCH1 and the impact of HH agonists and antagonists on NPC specification. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation.
Subject(s)
Hedgehog Proteins , Organoids , Signal Transduction , Spinal Cord , Hedgehog Proteins/metabolism , Hedgehog Proteins/genetics , Animals , Organoids/metabolism , Organoids/cytology , Spinal Cord/metabolism , Spinal Cord/cytology , Mice , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Patched-1 Receptor/metabolism , Patched-1 Receptor/geneticsABSTRACT
BACKGROUND: Sonic Hedgehog (SHH) is a fundamental signaling pathway that controls tissue reconstruction, stem cell biology, and differentiation and has a role in gut tissue homeostasis and development. Dysregulation of SHH leads to the development of HCC. METHODS, AND RESULTS: The present study was conducted to compare the effects of mesenchymal stem cells (MSCs) and curcumin on SHH molecular targets in an experimental model of HCC in rats. One hundred rats were divided equally into the following groups: control group, HCC group, HCC group received MSCs, HCC group received curcumin, and HCC group received MSCs and curcumin. Histopathological examinations were performed, and gene expression of SHH signaling target genes (SHH, PTCH1, SMOH, and GLI1) was assessed by real-time PCR in rat liver tissue. Results showed that SHH target genes were significantly upregulated in HCC-untreated rat groups and in MSC-treated groups, with no significant difference between them. Administration of curcumin with or without combined administration of MSCs led to a significant down-regulation of SHH target genes, with no significant differences between both groups. As regards the histopathological examination of liver tissues, both curcumin and MSCs, either through separate use or their combined use, led to a significant restoration of normal liver pathology. CONCLUSIONS: In conclusion, SHH signaling is upregulated in the HCC experimental model. MSCs do not inhibit the upregulated SHH target genes in HCC. Curcumin use with or without MSCs administration led to a significant down-regulation of SHH signaling in HCC and a significant restoration of normal liver pathology.