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1.
Cell ; 187(2): 276-293.e23, 2024 01 18.
Article in English | MEDLINE | ID: mdl-38171360

ABSTRACT

During development, morphogens pattern tissues by instructing cell fate across long distances. Directly visualizing morphogen transport in situ has been inaccessible, so the molecular mechanisms ensuring successful morphogen delivery remain unclear. To tackle this longstanding problem, we developed a mouse model for compromised sonic hedgehog (SHH) morphogen delivery and discovered that endocytic recycling promotes SHH loading into signaling filopodia called cytonemes. We optimized methods to preserve in vivo cytonemes for advanced microscopy and show endogenous SHH localized to cytonemes in developing mouse neural tubes. Depletion of SHH from neural tube cytonemes alters neuronal cell fates and compromises neurodevelopment. Mutation of the filopodial motor myosin 10 (MYO10) reduces cytoneme length and density, which corrupts neuronal signaling activity of both SHH and WNT. Combined, these results demonstrate that cytoneme-based signal transport provides essential contributions to morphogen dispersion during mammalian tissue development and suggest MYO10 is a key regulator of cytoneme function.


Subject(s)
Cell Membrane Structures , Myosins , Neural Tube , Signal Transduction , Animals , Mice , Biological Transport , Cell Membrane Structures/metabolism , Hedgehog Proteins/metabolism , Myosins/metabolism , Pseudopodia/metabolism , Neural Tube/cytology , Neural Tube/metabolism
2.
Cell ; 180(6): 1262-1271.e15, 2020 03 19.
Article in English | MEDLINE | ID: mdl-32169219

ABSTRACT

Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.


Subject(s)
Enhancer Elements, Genetic/genetics , High-Throughput Screening Assays/methods , Polydactyly/genetics , Animals , Enhancer Elements, Genetic/physiology , Gene Expression Regulation, Developmental/genetics , Gene Knock-In Techniques/methods , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Humans , Mice , Mutation , Phenotype , Polydactyly/metabolism , RNA, Untranslated/genetics
3.
Cell ; 169(7): 1327-1341.e23, 2017 Jun 15.
Article in English | MEDLINE | ID: mdl-28622513

ABSTRACT

Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole-exome sequencing and DNA copy number analyses, and we analyzed 196 HCC cases by DNA methylation, RNA, miRNA, and proteomic expression also. DNA sequencing and mutation analysis identified significantly mutated genes, including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or downregulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.


Subject(s)
Carcinoma, Hepatocellular/genetics , Genomics , Liver Neoplasms/genetics , Carcinoma, Hepatocellular/virology , DNA Methylation , Humans , Isocitrate Dehydrogenase/genetics , Liver Neoplasms/virology , MicroRNAs/genetics , Mutation
4.
Cell ; 167(3): 633-642.e11, 2016 Oct 20.
Article in English | MEDLINE | ID: mdl-27768887

ABSTRACT

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.


Subject(s)
Biological Evolution , Enhancer Elements, Genetic , Extremities/growth & development , Hedgehog Proteins/genetics , Snakes/genetics , Animals , Base Sequence , Evolution, Molecular , Gene Knock-In Techniques , Mice , Mice, Transgenic , Mutation , Phylogeny , Snakes/classification
5.
Mol Cell ; 81(24): 5025-5038.e10, 2021 12 16.
Article in English | MEDLINE | ID: mdl-34890564

ABSTRACT

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.


Subject(s)
Acyltransferases/antagonists & inhibitors , Acyltransferases/metabolism , Enzyme Inhibitors/pharmacology , Hedgehog Proteins/metabolism , Membrane Proteins/metabolism , Acylation , Acyltransferases/genetics , Acyltransferases/ultrastructure , Allosteric Regulation , Animals , COS Cells , Catalytic Domain , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Heme/metabolism , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Molecular Dynamics Simulation , Palmitoyl Coenzyme A/metabolism , Protein Conformation , Signal Transduction , Structure-Activity Relationship
6.
Development ; 151(14)2024 Jul 15.
Article in English | MEDLINE | ID: mdl-38940473

ABSTRACT

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/metabolism
7.
Development ; 2024 Aug 19.
Article in English | MEDLINE | ID: mdl-39157903

ABSTRACT

Ciliopathies are characterized by the absence or dysfunction of primary cilia. Despite the fact that cognitive impairments are a common feature of ciliopathies, how cilia dysfunction affects neuronal development has not been characterized in detail. Here, we show that primary cilium-mediated signaling is required cell-autonomously by neurons during neural circuit formation. In particular, a functional primary cilium is crucial during axonal pathfinding for the switch in responsiveness of axons at a choice point, or intermediate target. Utilizing different animal models and in vivo, ex vivo, as well as in vitro experiments, we provide evidence for a critical role of primary cilium-mediated signaling in long-range axon guidance. The primary cilium on the cell body of commissural neurons transduces long-range guidance signals sensed by growth cones navigating an intermediate target. In extension of our finding that Shh is required for the rostral turn of post-crossing commissural axons, we suggest a model implicating the primary cilium in Shh signaling upstream of a transcriptional change of axon guidance receptors, which in turn mediate the repulsive response to floorplate-derived Shh shown by post-crossing commissural axons.

8.
Mol Cell ; 76(3): 473-484.e7, 2019 11 07.
Article in English | MEDLINE | ID: mdl-31494034

ABSTRACT

Enhancers can regulate the promoters of their target genes over very large genomic distances. It is widely assumed that mechanisms of enhancer action involve the reorganization of three-dimensional chromatin architecture, but this is poorly understood. The predominant model involves physical enhancer-promoter interaction by looping out the intervening chromatin. However, studying the enhancer-driven activation of the Sonic hedgehog gene (Shh), we have identified a change in chromosome conformation that is incompatible with this simple looping model. Using super-resolution 3D-FISH and chromosome conformation capture, we observe a decreased spatial proximity between Shh and its enhancers during the differentiation of embryonic stem cells to neural progenitors. We show that this can be recapitulated by synthetic enhancer activation, is impeded by chromatin-bound proteins located between the enhancer and the promoter, and appears to involve the catalytic activity of poly (ADP-ribose) polymerase. Our data suggest that models of enhancer-promoter communication need to encompass chromatin conformations other than looping.


Subject(s)
Chromatin Assembly and Disassembly , Enhancer Elements, Genetic , Hedgehog Proteins/metabolism , Mouse Embryonic Stem Cells/metabolism , Neurogenesis , Neurons/metabolism , Promoter Regions, Genetic , Transcriptional Activation , Animals , Cell Line , Gene Expression Regulation, Developmental , Hedgehog Proteins/genetics , Mice , Models, Genetic , Neurogenesis/genetics , Nucleic Acid Conformation , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism
9.
EMBO J ; 41(1): e108843, 2022 01 04.
Article in English | MEDLINE | ID: mdl-34981518

ABSTRACT

Primary cilia are antenna-like organelles required for signalling transduction. How cilia structure is mechanistically maintained at steady-state to promote signalling is largely unknown. Here, we define that mammalian primary cilia axonemes are formed by proximal segment (PS) and distal segment (DS) delineated by tubulin polyglutamylation-rich and -poor regions, respectively. The analysis of proximal/distal segmentation indicated that perturbations leading to cilia over-elongation influenced PS or DS length with a different impact on cilia behaviour. We identified septins as novel repressors of DS growth. We show that septins control the localisation of MKS3 and CEP290 required for a functional transition zone (TZ), and the cilia tip accumulation of the microtubule-capping kinesin KIF7, a cilia-growth inhibitor. Live-cell imaging and analysis of sonic-hedgehog (SHH) signalling activation established that DS over-extension increased cilia ectocytosis events and decreased SHH activation. Our data underlines the importance of understanding cilia segmentation for length control and cilia-dependent signalling.


Subject(s)
Cilia/metabolism , Septins/metabolism , Animals , Antigens, Neoplasm/metabolism , Cell Cycle Proteins/metabolism , Cell Line , Cytoskeletal Proteins/metabolism , Hedgehog Proteins/metabolism , Humans , Kinesins/metabolism , Membrane Proteins/metabolism , Mice , Microtubules/metabolism , Retinal Pigment Epithelium/cytology , Signal Transduction
10.
Development ; 150(7)2023 04 01.
Article in English | MEDLINE | ID: mdl-36971701

ABSTRACT

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.


Subject(s)
Hedgehog Proteins , Signal Transduction , Mice , Animals , Hedgehog Proteins/metabolism , Signal Transduction/genetics , Mouth , Incisor/metabolism
11.
Development ; 150(13)2023 07 01.
Article in English | MEDLINE | ID: mdl-37381820

ABSTRACT

Cerebellar granule neurons (CGNs) are the most abundant neurons in the human brain. Dysregulation of their development underlies movement disorders and medulloblastomas. It is suspected that these disorders arise in progenitor states of the CGN lineage, for which human models are lacking. Here, we have differentiated human hindbrain neuroepithelial stem (hbNES) cells to CGNs in vitro using soluble growth factors, recapitulating key progenitor states in the lineage. We show that hbNES cells are not lineage committed and retain rhombomere 1 regional identity. Upon differentiation, hbNES cells transit through a rhombic lip (RL) progenitor state at day 7, demonstrating human specific sub-ventricular cell identities. This RL state is followed by an ATOH1+ CGN progenitor state at day 14. By the end of a 56-day differentiation procedure, we obtain functional neurons expressing CGN markers GABAARα6 and vGLUT2. We show that sonic hedgehog promotes GABAergic lineage specification and CGN progenitor proliferation. Our work presents a new model with which to study development and diseases of the CGN lineage in a human context.


Subject(s)
Cerebellum , Hedgehog Proteins , Humans , Hedgehog Proteins/metabolism , Rhombencephalon/metabolism , Cell Differentiation/physiology , Neurogenesis , Stem Cells
12.
Proc Natl Acad Sci U S A ; 120(4): e2209964120, 2023 Jan 24.
Article in English | MEDLINE | ID: mdl-36669111

ABSTRACT

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B, which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.


Subject(s)
Ferrets , Hedgehog Proteins , Animals , Female , Humans , Mice , Pregnancy , Central Nervous System/metabolism , Cilia/genetics , Cilia/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Mice, Knockout , Signal Transduction
13.
Proc Natl Acad Sci U S A ; 120(23): e2220037120, 2023 06 06.
Article in English | MEDLINE | ID: mdl-37252980

ABSTRACT

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.


Subject(s)
Calcium , Hedgehog Proteins , Xenopus Proteins , Calcium/metabolism , Cell Differentiation , Cilia/metabolism , Hedgehog Proteins/metabolism , Neural Tube/metabolism , Neurogenesis/physiology , Xenopus laevis , Animals
14.
Dev Biol ; 516: 138-147, 2024 Aug 21.
Article in English | MEDLINE | ID: mdl-39173434

ABSTRACT

The teleost Astyanax mexicanus consists of surface dwelling (surface fish) and cave dwelling (cavefish) forms. Cavefish have evolved in subterranean habitats characterized by reduced oxygen levels (hypoxia) and exhibit a subset of phenotypic traits controlled by increased Sonic hedgehog (Shh) signaling along the embryonic midline. The enhancement of primitive hematopoietic domains, which are formed bilaterally in the anterior and posterior lateral plate mesoderm, are responsible for the development of more larval erythrocytes in cavefish relative to surface fish. In this study, we determine the role of hypoxia and Shh signaling in the development and evolution of primitive hematopoiesis in cavefish. We show that hypoxia treatment during embryogenesis increases primitive hematopoiesis and erythrocyte development in surface fish. We also demonstrate that upregulation of Shh midline signaling by the Smoothened agonist SAG increases primitive hematopoiesis and erythrocyte development in surface fish, whereas Shh downregulation via treatment with the Smoothened inhibitor cyclopamine decreases these traits in cavefish. Together these results suggest that hematopoietic enhancement is regulated by hypoxia and Shh signaling. Lastly, we demonstrate that hypoxia enhances expression of Shh signaling along the midline of surface fish embryos. We conclude that hypoxia-mediated Shh plasticity may be a driving force for the adaptive evolution of primitive hematopoiesis and erythrocyte development in cavefish.

15.
Development ; 149(3)2022 02 01.
Article in English | MEDLINE | ID: mdl-35112129

ABSTRACT

The tracheal epithelium is a primary target for pulmonary diseases as it provides a conduit for air flow between the environment and the lung lobes. The cellular and molecular mechanisms underlying airway epithelial cell proliferation and differentiation remain poorly understood. Hedgehog (HH) signaling orchestrates communication between epithelial and mesenchymal cells in the lung, where it modulates stromal cell proliferation, differentiation and signaling back to the epithelium. Here, we reveal a previously unreported autocrine function of HH signaling in airway epithelial cells. Epithelial cell depletion of the ligand sonic hedgehog (SHH) or its effector smoothened (SMO) causes defects in both epithelial cell proliferation and differentiation. In cultured primary human airway epithelial cells, HH signaling inhibition also hampers cell proliferation and differentiation. Epithelial HH function is mediated, at least in part, through transcriptional activation, as HH signaling inhibition leads to downregulation of cell type-specific transcription factor genes in both the mouse trachea and human airway epithelial cells. These results provide new insights into the role of HH signaling in epithelial cell proliferation and differentiation during airway development.


Subject(s)
Autocrine Communication/physiology , Cell Differentiation , Cell Proliferation , Hedgehog Proteins/metabolism , Signal Transduction/genetics , Animals , Cells, Cultured , Down-Regulation , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Hedgehog Proteins/deficiency , Hedgehog Proteins/genetics , Humans , Lung/growth & development , Lung/metabolism , Lung/pathology , Mice , Mice, Knockout , Smoothened Receptor/deficiency , Smoothened Receptor/genetics , Smoothened Receptor/metabolism , Trachea/cytology , Trachea/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
16.
J Pathol ; 262(4): 427-440, 2024 04.
Article in English | MEDLINE | ID: mdl-38229567

ABSTRACT

Radiotherapy is one of the standard therapeutic regimens for medulloblastoma (MB). Tumor cells utilize DNA damage repair (DDR) mechanisms to survive and develop resistance during radiotherapy. It has been found that targeting DDR sensitizes tumor cells to radiotherapy in several types of cancer, but whether and how DDR pathways are involved in the MB radiotherapy response remain to be determined. Single-cell RNA sequencing was carried out on 38 MB tissues, followed by expression enrichment assays. Fanconi anemia group D2 gene (FANCD2) expression was evaluated in MB samples and public MB databases. The function of FANCD2 in MB cells was examined using cell counting assays (CCK-8), clone formation, lactate dehydrogenase activity, and in mouse orthotopic models. The FANCD2-related signaling pathway was investigated using assays of peroxidation, a malondialdehyde assay, a reduced glutathione assay, and using FerroOrange to assess intracellular iron ions (Fe2+ ). Here, we report that FANCD2 was highly expressed in the malignant sonic hedgehog (SHH) MB subtype (SHH-MB). FANCD2 played an oncogenic role and predicted worse prognosis in SHH-MB patients. Moreover, FANCD2 knockdown markedly suppressed viability, mobility, and growth of SHH-MB cells and sensitized SHH-MB cells to irradiation. Mechanistically, FANCD2 deficiency led to an accumulation of Fe2+ due to increased divalent metal transporter 1 expression and impaired glutathione peroxidase 4 activity, which further activated ferroptosis and reduced proliferation of SHH-MB cells. Using an orthotopic mouse model, we observed that radiotherapy combined with silencing FANCD2 significantly inhibited the growth of SHH-MB cell-derived tumors in vivo. Our study revealed FANCD2 as a potential therapeutic target in SHH-MB and silencing FANCD2 could sensitize SHH-MB cells to radiotherapy via inducing ferroptosis. © 2024 The Pathological Society of Great Britain and Ireland.


Subject(s)
Cerebellar Neoplasms , Fanconi Anemia , Ferroptosis , Medulloblastoma , Mice , Animals , Humans , Medulloblastoma/genetics , Medulloblastoma/radiotherapy , Ferroptosis/genetics , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/radiotherapy , Cell Line, Tumor , Fanconi Anemia Complementation Group D2 Protein/genetics
17.
Semin Cell Dev Biol ; 129: 115-125, 2022 09.
Article in English | MEDLINE | ID: mdl-35466055

ABSTRACT

The primary cilium is a hair-like sensory compartment that protrudes from the cellular surface. The primary cilium is enriched in a variety of signaling molecules that regulate cellular activities. Stem cells have primary cilia. They reside in a specialized environment, called the stem cell niche. This niche contains a variety of secreted factors, and some of their receptors are localized in the primary cilia of stem cells. Here, we summarize the current understanding of the function of cilia in compartmentalized signaling in stem cells. We describe how ciliary signaling regulates stem cells and progenitor cells during development, tissue homeostasis and tumorigenesis. We summarize our understanding of cilia regulated signaling -primary involving the hedgehog pathway- in stem cells in diverse settings that include neuroepithelial cells, radial glia, cerebellar granule neuron precursors, hematopoietic stem cells, hair follicle stem cells, bone marrow mesenchymal stem cells and mammary gland stem cells. Overall, our review highlights a variety of roles that ciliary signaling plays in regulating stem cells throughout life.


Subject(s)
Hedgehog Proteins , Receptors, G-Protein-Coupled , Cilia/metabolism , Hedgehog Proteins/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiology , Stem Cells/metabolism
18.
Dev Biol ; 504: 58-74, 2023 12.
Article in English | MEDLINE | ID: mdl-37739118

ABSTRACT

The Sonic hedgehog (SHh) signaling pathway is an imperative operating network that helps in regulates the critical events during the development processes like multicellular embryo growth and patterning. Disruptions in SHh pathway regulation can have severe consequences, including congenital disabilities, stem cell renewal, tissue regeneration, and cancer/tumor growth. Activation of the SHh signal occurs when SHh binds to the receptor complex of Patch (Ptc)-mediated Smoothened (Smo) (Ptc-smo), initiating downstream signaling. This review explores how pharmacological modulation of the SHh pathway affects angiogenesis through canonical and non-canonical pathways. The canonical pathway for angiogenesis involves the activation of angiogenic cytokines such as fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), placental growth factor (PGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), stromal cell-derived factor 1α, transforming growth factor-ß1 (TGF-ß1), and angiopoietins (Ang-1 and Ang-2), which facilitate the process of angiogenesis. The Non-canonical pathway includes indirect activation of certain pathways like iNOS/Netrin-1/PKC, RhoA/Rock, ERK/MAPK, PI3K/Akt, Wnt/ß-catenin, Notch signaling pathway, and so on. This review will provide a better grasp of the mechanistic approach of SHh in mediating angiogenesis, which can aid in the suppression of certain cancer and tumor growths.


Subject(s)
Hedgehog Proteins , Neoplasms , Female , Humans , Hedgehog Proteins/metabolism , Vascular Endothelial Growth Factor A/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Placenta Growth Factor , Signal Transduction/physiology
19.
Glia ; 72(8): 1469-1483, 2024 08.
Article in English | MEDLINE | ID: mdl-38771121

ABSTRACT

Myelination is the terminal step in a complex and precisely timed program that orchestrates the proliferation, migration and differentiation of oligodendroglial cells. It is thought that Sonic Hedgehog (Shh) acting on Smoothened (Smo) participates in regulating this process, but that these effects are highly context dependent. Here, we investigate oligodendroglial development and remyelination from three specific transgenic lines: NG2-CreERT2 (control), Smofl/fl/NG2-CreERT2 (loss of function), and SmoM2/NG2-CreERT2 (gain of function), as well as pharmacological manipulation that enhance or inhibit the Smo pathway (Smoothened Agonist (SAG) or cyclopamine treatment, respectively). To explore the effects of Shh/Smo on differentiation and myelination in vivo, we developed a highly quantifiable model by transplanting oligodendrocyte precursor cells (OPCs) in the retina. We find that myelination is greatly enhanced upon cyclopamine treatment and hypothesize that Shh/Smo could promote OPC proliferation to subsequently inhibit differentiation. Consistent with this hypothesis, we find that the genetic activation of Smo significantly increased numbers of OPCs and decreased oligodendrocyte differentiation when we examined the corpus callosum during development and after cuprizone demyelination and remyelination. However, upon loss of function with the conditional ablation of Smo, myelination in the same scenarios are unchanged. Taken together, our present findings suggest that the Shh pathway is sufficient to maintain OPCs in an undifferentiated state, but is not necessary for myelination and remyelination.


Subject(s)
Cell Differentiation , Hedgehog Proteins , Mice, Transgenic , Myelin Sheath , Oligodendrocyte Precursor Cells , Smoothened Receptor , Animals , Hedgehog Proteins/metabolism , Oligodendrocyte Precursor Cells/metabolism , Oligodendrocyte Precursor Cells/drug effects , Smoothened Receptor/metabolism , Smoothened Receptor/genetics , Myelin Sheath/metabolism , Cell Differentiation/physiology , Cell Differentiation/drug effects , Veratrum Alkaloids/pharmacology , Mice , Remyelination/physiology , Remyelination/drug effects , Oligodendroglia/metabolism , Oligodendroglia/drug effects , Oligodendroglia/physiology , Mice, Inbred C57BL , Signal Transduction/physiology , Signal Transduction/drug effects
20.
J Cell Biochem ; : e30637, 2024 Aug 16.
Article in English | MEDLINE | ID: mdl-39150066

ABSTRACT

Glioblastoma (GBM) aggressiveness is partly driven by the reactivation of signaling pathways such as Sonic hedgehog (SHH) and the interaction with its microenvironment. SHH pathway activation is one of the phenomena behind the glial transformation in response to tumor growth. The reactivation of the SHH signaling cascade during GBM-astrocyte interaction is highly relevant to understanding the mechanisms used by the tumor to modulate the adjacent stroma. The role of reactive astrocytes considering SHH signaling during GBM progression is investigated using a 3D in vitro model. T98G GBM spheroids displayed significant downregulation of SHH (61.4 ± 9.3%), GLI-1 (6.5 ± 3.7%), Ki-67 (33.7 ± 8.1%), and mutant MTp53 (21.3 ± 10.6%) compared to the CONTROL group when incubated with conditioned medium of reactive astrocytes (CM-AST). The SHH pathway inhibitor, GANT-61, significantly reduced previous markers (SHH = 43.0 ± 12.1%; GLI-1 = 9.5 ± 3.4%; Ki-67 = 31.9 ± 4.6%; MTp53 = 6.5 ± 7.5%) compared to the CONTROL, and a synergistic effect could be observed between GANT-61 and CM-AST. The volume (2.0 ± 0.2 × 107 µm³), cell viability (80.4 ± 3.2%), and migration (41 ± 10%) of GBM spheroids were significantly reduced in the presence of GANT-61 and CM-AST when compared to CM-AST after 72 h (volume = 2.3 ± 0.4 × 107 µm³; viability = 92.2 ± 6.5%; migration = 102.5 ± 14.6%). Results demonstrated that factors released by reactive astrocytes promoted a neuroprotective effect preventing GBM progression using a 3D in vitro model potentiated by SHH pathway inhibition.

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