RESUMO
O-GlcNAc is a dynamic post-translational modification (PTM) that regulates protein functions. In studying the regulatory roles of O-GlcNAc, a major roadblock is the inability to change O-GlcNAcylation on a single protein at a time. Herein, we developed a dual RNA-aptamer-based approach that simultaneously targeted O-GlcNAc transferase (OGT) and ß-catenin, the key transcription factor of the Wnt signaling pathway, to selectively increase O-GlcNAcylation of the latter without affecting other OGT substrates. Using the OGT/ß-catenin dual-specificity aptamers, we found that O-GlcNAcylation of ß-catenin stabilizes the protein by inhibiting its interaction with ß-TrCP. O-GlcNAc also increases ß-catenin's interaction with EZH2, recruits EZH2 to promoters, and dramatically alters the transcriptome. Further, by coupling riboswitches or an inducible expression system to aptamers, we enabled inducible regulation of protein-specific O-GlcNAcylation. Together, our findings demonstrate the efficacy and versatility of dual-specificity aptamers for regulating O-GlcNAcylation on individual proteins.
Assuntos
Aptâmeros de Nucleotídeos , beta Catenina/metabolismo , Processamento de Proteína Pós-Traducional , Via de Sinalização Wnt , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismoRESUMO
Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Animais , Dissulfetos/metabolismo , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Receptores Acoplados a Proteínas G/isolamento & purificação , Receptores Acoplados a Proteínas G/ultraestrutura , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Proteínas Wnt/química , Proteínas Wnt/isolamento & purificação , Proteínas Wnt/ultraestruturaRESUMO
The majority of animal phyla have species that can regenerate. Comparing regeneration across animals can reconstruct the molecular and cellular evolutionary history of this process. Recent studies have revealed some similarity in regeneration mechanisms, but rigorous comparative methods are needed to assess whether these resemblances are ancestral pathways (homology) or are the result of convergent evolution (homoplasy). This review aims to provide a framework for comparing regeneration across animals, focusing on gene regulatory networks (GRNs), which are substrates for assessing process homology. The homology of the wound-induced activation of Wnt signaling and of adult stem cells provides examples of ongoing studies of regeneration that enable comparisons in a GRN framework. Expanding the study of regeneration GRNs in currently studied species and broadening taxonomic sampling for these approaches will identify processes that are unifying principles of regeneration biology across animals. These insights are important both for evolutionary studies of regeneration and for human regenerative medicine.
Assuntos
Células-Tronco Adultas , Redes Reguladoras de Genes , Animais , Redes Reguladoras de Genes/genética , Via de Sinalização Wnt/genéticaRESUMO
The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Análise de Célula Única , Via de Sinalização WntRESUMO
The function of somatic stem cells declines with age. Understanding the molecular underpinnings of this decline is key to counteract age-related disease. Here, we report a dramatic drop in the neural stem cells (NSCs) number in the aging murine brain. We find that this smaller stem cell reservoir is protected from full depletion by an increase in quiescence that makes old NSCs more resistant to regenerate the injured brain. Once activated, however, young and old NSCs show similar proliferation and differentiation capacity. Single-cell transcriptomics of NSCs indicate that aging changes NSCs minimally. In the aging brain, niche-derived inflammatory signals and the Wnt antagonist sFRP5 induce quiescence. Indeed, intervention to neutralize them increases activation of old NSCs during homeostasis and following injury. Our study identifies quiescence as a key feature of old NSCs imposed by the niche and uncovers ways to activate NSCs to repair the aging brain.
Assuntos
Encéfalo/fisiologia , Fatores Etários , Animais , Encéfalo/citologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Homeostase , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Neurogênese , Nicho de Células-TroncoRESUMO
Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.
Assuntos
Adenocarcinoma/patologia , Organoides/patologia , Neoplasias Gástricas/patologia , Estômago/patologia , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Carcinogênese , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Organoides/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Trombospondinas/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The mitochondrial unfolded protein response (UPRmt) can be triggered in a cell-non-autonomous fashion across multiple tissues in response to mitochondrial dysfunction. The ability to communicate information about the presence of mitochondrial stress enables a global response that can ultimately better protect an organism from local mitochondrial challenges. We find that animals use retromer-dependent Wnt signaling to propagate mitochondrial stress signals from the nervous system to peripheral tissues. Specifically, the polyQ40-triggered activation of mitochondrial stress or reduction of cco-1 (complex IV subunit) in neurons of C. elegans results in the Wnt-dependent induction of cell-non-autonomous UPRmt in peripheral cells. Loss-of-function mutations of retromer complex components that are responsible for recycling the Wnt secretion-factor/MIG-14 prevent Wnt secretion and thereby suppress cell-non-autonomous UPRmt. Neuronal expression of the Wnt ligand/EGL-20 is sufficient to induce cell-non-autonomous UPRmt in a retromer complex-, Wnt signaling-, and serotonin-dependent manner, clearly implicating Wnt signaling as a strong candidate for the "mitokine" signal.
Assuntos
Animais Geneticamente Modificados/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mitocôndrias/metabolismo , Poliubiquitina/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Proteínas Wnt/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/genética , Neurônios/citologia , Neurônios/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas Wnt/genéticaRESUMO
Neural induction in vertebrates generates a CNS that extends the rostral-caudal length of the body. The prevailing view is that neural cells are initially induced with anterior (forebrain) identity; caudalizing signals then convert a proportion to posterior fates (spinal cord). To test this model, we used chromatin accessibility to define how cells adopt region-specific neural fates. Together with genetic and biochemical perturbations, this identified a developmental time window in which genome-wide chromatin-remodeling events preconfigure epiblast cells for neural induction. Contrary to the established model, this revealed that cells commit to a regional identity before acquiring neural identity. This "primary regionalization" allocates cells to anterior or posterior regions of the nervous system, explaining how cranial and spinal neurons are generated at appropriate axial positions. These findings prompt a revision to models of neural induction and support the proposed dual evolutionary origin of the vertebrate CNS.
Assuntos
Montagem e Desmontagem da Cromatina , Indução Embrionária , Neurogênese , Animais , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Medula Espinal/metabolismoRESUMO
The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.
Assuntos
Pulmão/citologia , Mesoderma/citologia , Animais , Homeostase , Pulmão/fisiologia , Camundongos , Organoides/citologia , Alvéolos Pulmonares/citologia , Receptores Acoplados a Proteínas G/análise , Análise de Sequência de RNA , Análise de Célula Única , Transcrição GênicaRESUMO
Intestinal stem cell maturation and development coincide with gut microbiota exposure after birth. Here, we investigated how early life microbial exposure, and disruption of this process, impacts the intestinal stem cell niche and development. Single-cell transcriptional analysis revealed impaired stem cell differentiation into Paneth cells and macrophage specification upon antibiotic treatment in early life. Mouse genetic and organoid co-culture experiments demonstrated that a CD206+ subset of intestinal macrophages secreted Wnt ligands, which maintained the mesenchymal niche cells important for Paneth cell differentiation. Antibiotics and reduced numbers of Paneth cells are associated with the deadly infant disease, necrotizing enterocolitis (NEC). We showed that colonization with Lactobacillus or transfer of CD206+ macrophages promoted Paneth cell differentiation and reduced NEC severity. Together, our work defines the gut microbiota-mediated regulation of stem cell niches during early postnatal development.
Assuntos
Enterocolite Necrosante , Microbioma Gastrointestinal , Camundongos , Animais , Celulas de Paneth/fisiologia , Diferenciação Celular/fisiologia , MacrófagosRESUMO
Biomolecular condensates have emerged as a major organizational principle in the cell. However, the formation, maintenance, and dissolution of condensates are still poorly understood. Transcriptional machinery partitions into biomolecular condensates at key cell identity genes to activate these. Here, we report a specific perturbation of WNT-activated ß-catenin condensates that disrupts oncogenic signaling. We use a live-cell condensate imaging method in human cancer cells to discover FOXO and TCF-derived peptides that specifically inhibit ß-catenin condensate formation on DNA, perturb nuclear ß-catenin condensates in cells, and inhibit ß-catenin-driven transcriptional activation and colorectal cancer cell growth. We show that these peptides compete with homotypic intermolecular interactions that normally drive condensate formation. Using this framework, we derive short peptides that specifically perturb condensates and transcriptional activation of YAP and TAZ in the Hippo pathway. We propose a "monomer saturation" model in which short interacting peptides can be used to specifically inhibit condensate-associated transcription in disease.
Assuntos
Neoplasias , beta Catenina , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Transdução de Sinais , Via de Sinalização Hippo , Peptídeos/genéticaRESUMO
Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease.
Assuntos
Moléculas de Adesão Celular/química , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/química , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Clonagem Molecular , Cristalografia por Raios X , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/antagonistas & inibidores , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores da Família Eph/antagonistas & inibidores , Receptores da Família Eph/química , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Spodoptera , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
The GPR124/RECK/WNT7 pathway is an essential regulator of CNS angiogenesis and blood-brain barrier (BBB) function. GPR124, a brain endothelial adhesion seven-pass transmembrane protein, associates with RECK, which binds and stabilizes newly synthesized WNT7 that is transferred to frizzled (FZD) to initiate canonical ß-catenin signaling. GPR124 remains enigmatic: although its extracellular domain (ECD) is essential, the poorly conserved intracellular domain (ICD) appears to be variably required in mammals versus zebrafish, potentially via adaptor protein bridging of GPR124 and FZD ICDs. GPR124 ICD deletion impairs zebrafish angiogenesis, but paradoxically retains WNT7 signaling upon mammalian transfection. We thus investigated GPR124 ICD function using the mouse deletion mutant Gpr124ΔC. Despite inefficiently expressed GPR124ΔC protein, Gpr124ΔC/ΔC mice could be born with normal cerebral cortex angiogenesis, in comparison with Gpr124-/- embryonic lethality, forebrain avascularity and hemorrhage. Gpr124ΔC/ΔC vascular phenotypes were restricted to sporadic ganglionic eminence angiogenic defects, attributable to impaired GPR124ΔC protein expression. Furthermore, Gpr124ΔC and the recombinant GPR124 ECD rescued WNT7 signaling in culture upon brain endothelial Gpr124 knockdown. Thus, in mice, GPR124-regulated CNS forebrain angiogenesis and BBB function are exerted by ICD-independent functionality, extending the signaling mechanisms used by adhesion seven-pass transmembrane receptors.
Assuntos
Barreira Hematoencefálica , Encéfalo , Neovascularização Fisiológica , Receptores Acoplados a Proteínas G , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/embriologia , Neovascularização Fisiológica/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Camundongos , Encéfalo/metabolismo , Encéfalo/embriologia , Domínios Proteicos , Camundongos Knockout , Transdução de Sinais , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Humanos , Células Endoteliais/metabolismo , Angiogênese , Proteínas Ligadas por GPIRESUMO
The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.
Assuntos
Proteínas de Ligação a DNA , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma , Fatores de Transcrição , Animais , Camundongos , Diferenciação Celular , Cromatina/metabolismo , Expressão Gênica , Mesoderma/metabolismo , Somitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The Wnt/ß-catenin signaling governs anterior-posterior neural patterning during development. Current human pluripotent stem cell (hPSC) differentiation protocols use a GSK3 inhibitor to activate Wnt signaling to promote posterior neural fate specification. However, GSK3 is a pleiotropic kinase involved in multiple signaling pathways and, as GSK3 inhibition occurs downstream in the signaling cascade, it bypasses potential opportunities for achieving specificity or regulation at the receptor level. Additionally, the specific roles of individual FZD receptors in anterior-posterior patterning are poorly understood. Here, we have characterized the cell surface expression of FZD receptors in neural progenitor cells with different regional identity. Our data reveal unique upregulation of FZD5 expression in anterior neural progenitors, and this expression is downregulated as cells adopt a posterior fate. This spatial regulation of FZD expression constitutes a previously unreported regulatory mechanism that adjusts the levels of ß-catenin signaling along the anterior-posterior axis and possibly contributes to midbrain-hindbrain boundary formation. Stimulation of Wnt/ß-catenin signaling in hPSCs, using a tetravalent antibody that selectively triggers FZD5 and LRP6 clustering, leads to midbrain progenitor differentiation and gives rise to functional dopaminergic neurons in vitro and in vivo.
Assuntos
Receptores Frizzled , Quinase 3 da Glicogênio Sintase , beta Catenina , Humanos , beta Catenina/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Mesencéfalo , Sistema Nervoso/metabolismo , Via de Sinalização Wnt , Animais , RatosRESUMO
Spatiotemporal control of Wnt signaling is essential for the development and homeostasis of many tissues. The transmembrane E3 ubiquitin ligases ZNRF3 (zinc and ring finger 3) and RNF43 (ring finger protein 43) antagonize Wnt signaling by promoting degradation of frizzled receptors. ZNRF3 and RNF43 are frequently inactivated in human cancer, but the molecular and therapeutic implications remain unclear. Here, we demonstrate that adrenocortical-specific loss of ZNRF3, but not RNF43, results in adrenal hyperplasia that depends on Porcupine-mediated Wnt ligand secretion. Furthermore, we discovered a Wnt/ß-catenin signaling gradient in the adrenal cortex that is disrupted upon loss of ZNRF3. Unlike ß-catenin gain-of-function models, which induce high Wnt/ß-catenin activation and expansion of the peripheral cortex, ZNRF3 loss triggers activation of moderate-level Wnt/ß-catenin signaling that drives proliferative expansion of only the histologically and functionally distinct inner cortex. Genetically reducing ß-catenin dosage significantly reverses the ZNRF3-deficient phenotype. Thus, homeostatic maintenance of the adrenal cortex is dependent on varying levels of Wnt/ß-catenin activation, which is regulated by ZNRF3.
Assuntos
Córtex Suprarrenal/metabolismo , Homeostase/genética , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Córtex Suprarrenal/citologia , Córtex Suprarrenal/crescimento & desenvolvimento , Doenças do Córtex Suprarrenal/fisiopatologia , Animais , Proliferação de Células/genética , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Modelos Animais , Ativação Transcricional/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Craniofacial morphogenesis requires complex interactions involving different tissues, signaling pathways, secreted factors and organelles. The details of these interactions remain elusive. In this study, we have analyzed the molecular mechanisms and homeostatic cellular activities governing soft palate development to improve regenerative strategies for individuals with cleft palate. We have identified canonical Wnt signaling as a key signaling pathway primarily active in cranial neural crest (CNC)-derived mesenchymal cells surrounding soft palatal myogenic cells. Using Osr2-Cre;ß-cateninfl/fl mice, we show that Wnt signaling is indispensable for mesenchymal cell proliferation and subsequently for myogenesis through mediating ciliogenesis. Specifically, we have identified that Wnt signaling directly regulates expression of the ciliary gene Ttll3. Impaired ciliary disassembly leads to differentiation defects in mesenchymal cells and indirectly disrupts myogenesis through decreased expression of Dlk1, a mesenchymal cell-derived pro-myogenesis factor. Moreover, we show that siRNA-mediated reduction of Ttll3 expression partly rescues mesenchymal cell proliferation and myogenesis in the palatal explant cultures from Osr2-Cre;ß-cateninfl/fl embryos. This study highlights the role of Wnt signaling in palatogenesis through the control of ciliary homeostasis, which establishes a new mechanism for Wnt-regulated craniofacial morphogenesis.
Assuntos
Fissura Palatina , Via de Sinalização Wnt , Camundongos , Animais , Via de Sinalização Wnt/fisiologia , Palato , Fissura Palatina/genética , Diferenciação Celular , Palato Mole , Homeostase , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Frizzled 2 (FZD2) is a transmembrane Wnt receptor. We previously identified a pathogenic human FZD2 variant in individuals with FZD2-associated autosomal dominant Robinow syndrome. The variant encoded a protein with a premature stop and loss of 17 amino acids, including a region of the consensus dishevelled-binding sequence. To model this variant, we used zygote microinjection and i-GONAD-based CRISPR/Cas9-mediated genome editing to generate a mouse allelic series. Embryos mosaic for humanized Fzd2W553* knock-in exhibited cleft palate and shortened limbs, consistent with patient phenotypes. We also generated two germline mouse alleles with small deletions: Fzd2D3 and Fzd2D4. Homozygotes for each allele exhibit a highly penetrant cleft palate phenotype, shortened limbs compared with wild type and perinatal lethality. Fzd2D4 craniofacial tissues indicated decreased canonical Wnt signaling. In utero treatment with IIIC3a (a DKK inhibitor) normalized the limb lengths in Fzd2D4 homozygotes. The in vivo replication represents an approach for further investigating the mechanism of FZD2 phenotypes and demonstrates the utility of CRISPR knock-in mice as a tool for investigating the pathogenicity of human genetic variants. We also present evidence for a potential therapeutic intervention.
Assuntos
Fissura Palatina , Nanismo , Deformidades Congênitas dos Membros , Anormalidades Urogenitais , Animais , Humanos , Camundongos , Fissura Palatina/genética , Nanismo/genética , Deformidades Congênitas dos Membros/genética , Anormalidades Urogenitais/genética , Via de Sinalização Wnt/genética , Modelos Animais de Doenças , Receptores Frizzled/genética , Técnicas de Introdução de GenesRESUMO
In embryonic stem cell (ESC) models for early development, spatially and temporally varying patterns of signaling and cell types emerge spontaneously. However, mechanistic insight into this dynamic self-organization is limited by a lack of methods for spatiotemporal control of signaling, and the relevance of signal dynamics and cell-to-cell variability to pattern emergence remains unknown. Here, we combine optogenetic stimulation, imaging and transcriptomic approaches to study self-organization of human ESCs (hESC) in two-dimensional (2D) culture. Morphogen dynamics were controlled via optogenetic activation of canonical Wnt/ß-catenin signaling (optoWnt), which drove broad transcriptional changes and mesendoderm differentiation at high efficiency (>99% cells). When activated within cell subpopulations, optoWnt induced cell self-organization into distinct epithelial and mesenchymal domains, mediated by changes in cell migration, an epithelial to mesenchymal-like transition and TGFß signaling. Furthermore, we demonstrate that such optogenetic control of cell subpopulations can be used to uncover signaling feedback mechanisms between neighboring cell types. These findings reveal that cell-to-cell variability in Wnt signaling is sufficient to generate tissue-scale patterning and establish a hESC model system for investigating feedback mechanisms relevant to early human embryogenesis.
Assuntos
Células-Tronco Pluripotentes , Via de Sinalização Wnt , Humanos , Via de Sinalização Wnt/genética , Optogenética , beta Catenina/metabolismo , Células-Tronco Embrionárias , Diferenciação Celular/genéticaRESUMO
Cardiac lineage specification in the mouse is controlled by TGFß and WNT signaling. From fly to fish, BMP has been identified as an indispensable heart inducer. A detailed analysis of the role of Bmp4 and its effectors Smad1/5, however, was still missing. We show that Bmp4 induces cardiac mesoderm formation in murine embryonic stem cells in vitro. Bmp4 first activates Wnt3 and upregulates Nodal. pSmad1/5 and the WNT effector Tcf3 form a complex, and together with pSmad2/3 activate mesoderm enhancers and Eomes. They then cooperate with Eomes to consolidate the expression of many mesoderm factors, including T. Eomes and T form a positive- feedback loop and open additional enhancers regulating early mesoderm genes, including the transcription factor Mesp1, establishing the cardiac mesoderm lineage. In parallel, the neural fate is suppressed. Our data confirm the pivotal role of Bmp4 in cardiac mesoderm formation in the mouse. We describe in detail the consecutive and cooperative actions of three signaling pathways, BMP, WNT and Nodal, and their effector transcription factors, during cardiac mesoderm specification.