RESUMEN
Formation of the body of vertebrate embryos proceeds sequentially by posterior addition of tissues from the tail bud. Cells of the tail bud and the posterior presomitic mesoderm, which control posterior elongation1, exhibit a high level of aerobic glycolysis that is reminiscent of the metabolic status of cancer cells experiencing the Warburg effect2,3. Glycolytic activity downstream of fibroblast growth factor controls WNT signalling in the tail bud3. In the neuromesodermal precursors of the tail bud4, WNT signalling promotes the mesodermal fate that is required for sustained axial elongation, at the expense of the neural fate3,5. How glycolysis regulates WNT signalling in the tail bud is currently unknown. Here we used chicken embryos and human tail bud-like cells differentiated in vitro from induced pluripotent stem cells to show that these cells exhibit an inverted pH gradient, with the extracellular pH lower than the intracellular pH, as observed in cancer cells6. Our data suggest that glycolysis increases extrusion of lactate coupled to protons via the monocarboxylate symporters. This contributes to elevating the intracellular pH in these cells, which creates a favourable chemical environment for non-enzymatic ß-catenin acetylation downstream of WNT signalling. As acetylated ß-catenin promotes mesodermal rather than neural fate7, this ultimately leads to activation of mesodermal transcriptional WNT targets and specification of the paraxial mesoderm in tail bud precursors. Our work supports the notion that some tumour cells reactivate a developmental metabolic programme.
Asunto(s)
Amnios/embriología , Glucólisis , Proteínas Wnt/metabolismo , Acetilación , Animales , Tipificación del Cuerpo , Embrión de Pollo , Humanos , Concentración de Iones de Hidrógeno , Ácido Láctico/metabolismo , Mesodermo/metabolismo , beta Catenina/metabolismoRESUMEN
CTR9 is one of five genes that form the PAF1 complex, which binds to RNA polymerase II and plays critical roles in transcriptional elongation and transcription-coupled histone modifications including histones H3K4me3 and H3K36me3. In this study, de novo CTR9 non-synonymous variants (p.(Glu15Asp) and p.(Pro25Arg)) were detected in two unrelated patients with macrocephaly, motor delay, and intellectual disability. A pull-down assay showed that the mutant CTR9 proteins had stronger affinities to the PAF1 protein than the wild-type protein. Functional analyses using zebrafish showed that the knockout of the ctr9 gene caused motor defects and enlargement of the telencephalon, which is homologous to the mammalian cerebrum. The rescue experiment, in which the human CTR9 mutants were introduced into ctr9-knockout zebrafish, failed to maintain the swimming posture of the ctr9-knockout fish, suggesting that the human CTR9 mutant proteins do not function normally in vivo. In addition, the overexpression of human CTR9 mutant mRNA caused telencephalon enlargement in zebrafish larvae, suggesting that the human CTR9 mutant proteins interfered with normal endogenous CTR9 function. We concluded that the two missense variants in CTR9 (p.(Glu15Asp) and p.(Pro25Arg)) cause a new syndrome involving macrocephaly, motor delay and intellectual disability through the loss of the normal function of CTR9 and the inhibition of the normal intrinsic CTR9 function of the contralateral allele.
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Discapacidad Intelectual , Megalencefalia , Animales , Humanos , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas Nucleares/genética , Discapacidad Intelectual/genética , Megalencefalia/genética , Proteínas Mutantes , Genética Humana , Mamíferos/metabolismo , Fosfoproteínas , Factores de TranscripciónRESUMEN
Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-ß pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper.
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Proteínas Tirosina Fosfatasas , Pez Cebra , Animales , Apoptosis/genética , Recuento de Células , Perros , Humanos , Hígado , Células de Riñón Canino Madin Darby , Proteínas de Neoplasias , Proteínas Tirosina Fosfatasas/genética , Pez Cebra/genéticaRESUMEN
Wnt2022 was held on November 15th-19th, 2022, in Awaji Yumebutai International Conference Center, Hyogo Prefecture, Japan, as an in-person meeting for the first time in last 3 years. Wnt signaling is a highly conserved pathway among various species. Since Wnt1 was discovered in 1982, a number of studies using many model animals and human samples have revealed that Wnt signaling plays crucial roles in embryonic development, tissue morphogenesis, and regeneration, as well as many other physiological and pathological processes. Since the year 2022 marks the 40th anniversary of Wnt research, we aimed to look back at our research progress and discuss the future direction of this field. The scientific program consisted of plenary lectures, invited talks, short talks selected from abstracts, and poster sessions. Whereas several different Wnt meetings have been held almost every year in Europe and the United States, this was the first Wnt meeting convened in Asia. Therefore, Wnt2022 was highly anticipated to bring together leaders and young scientists from Europe, the United States, and especially Asia and Oceania. In fact, 148 researchers from 21 countries attended this meeting. Although there were travel and administrative restrictions due to COVID-19, the meeting was highly successful in enabling face-to-face discussions.
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COVID-19 , Animales , Humanos , Asia , Japón , Vía de Señalización WntRESUMEN
Oncogenic mutations drive tumorigenesis, and single cells with oncogenic mutations act as the tumor seeds that gradually evolve into fully transformed tumors. However, oncogenic cell behavior and communication with neighboring cells during primary tumorigenesis remain poorly understood. We used the zebrafish, a small vertebrate model suitable for in vivo cell biology, to address these issues. We describe the cooperative and competitive communication between oncogenic cells and neighboring cells, as revealed by our recent zebrafish imaging studies. Newly generated oncogenic cells are actively eliminated by neighboring cells in healthy epithelia, whereas oncogenic cells cooperate with their neighbors to prime tumorigenesis in unhealthy epithelia via additional mutations or inflammation. In addition, we discuss the potential of zebrafish in vivo imaging to determine the initial steps of human tumorigenesis.Key words: zebrafish, imaging, cell-cell communication, cell competition, EDAC, senescence, primary tumorigenesis.
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Neoplasias , Pez Cebra , Animales , Humanos , Pez Cebra/genética , Transformación Celular Neoplásica/patología , Carcinogénesis , Neoplasias/patología , Comunicación CelularRESUMEN
Zebrafish is a useful model to study vertebrate hematopoiesis, but lack of antibodies to zebrafish proteins has limited purification of hematopoietic cells. Here, we purified neutrophils from larval and adult zebrafish using the lectin Phaseolus vulgaris erythroagglutinin (PHA-E) and DRAQ5, a DNA-staining fluorescent dye. In adult kidney marrow, we purified neutrophil-like PHA-E4low DRAQ5low cells, which neutrophil-type granules. Specifically, at 96-hr post-fertilization, we sorted large-sized cells from larvae using forward scatter and found that they consisted of PHA-Elow DRAQ5low populations. These cells had myeloperoxidase activity, were Sudan Black B-positive and expressed high levels of neutrophil-specific (csf3r and mpx) mRNAs, all neutrophil characteristics. Using this method, we conducted functional analysis suggesting that zyxin (Zyx) plays a role in neutrophil generation in zebrafish larvae. Overall, PHA-E and DRAQ5-based flow cytometry serves as a tool to purify zebrafish neutrophils.
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Citometría de Flujo/métodos , Hematopoyesis , Neutrófilos/citología , Cultivo Primario de Células/métodos , Animales , Células Cultivadas , Lectinas/metabolismo , Neutrófilos/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
PURPOSE: To expand the recent description of a new neurodevelopmental syndrome related to alterations in CDK19. METHODS: Individuals were identified through international collaboration. Functional studies included autophosphorylation assays for CDK19 Gly28Arg and Tyr32His variants and in vivo zebrafish assays of the CDK19G28R and CDK19Y32H. RESULTS: We describe 11 unrelated individuals (age range: 9 months to 14 years) with de novo missense variants mapped to the kinase domain of CDK19, including two recurrent changes at residues Tyr32 and Gly28. In vitro autophosphorylation and substrate phosphorylation assays revealed that kinase activity of protein was lower for p.Gly28Arg and higher for p.Tyr32His substitutions compared with that of the wild-type protein. Injection of CDK19 messenger RNA (mRNA) with either the Tyr32His or the Gly28Arg variants using in vivo zebrafish model significantly increased fraction of embryos with morphological abnormalities. Overall, the phenotype of the now 14 individuals with CDK19-related disorder includes universal developmental delay and facial dysmorphism, hypotonia (79%), seizures (64%), ophthalmologic anomalies (64%), and autism/autistic traits (56%). CONCLUSION: CDK19 de novo missense variants are responsible for a novel neurodevelopmental disorder. Both kinase assay and zebrafish experiments showed that the pathogenetic mechanism may be more diverse than previously thought.
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Discapacidad Intelectual , Trastornos del Neurodesarrollo , Animales , Quinasas Ciclina-Dependientes/genética , Mutación con Ganancia de Función , Humanos , Lactante , Mutación Missense , Pez Cebra/genéticaRESUMEN
CDK9 has been considered a candidate gene involved in the CHARGE-like syndrome in a pair of cousins. We report an 8-year-old boy with a strikingly similar phenotype including facial asymmetry, microtia with preauricular tags and bilateral hearing loss, cleft lip and palate, cardiac dysrhythmia, and undescended testes. Joint contracture, no finger flexion creases, and large halluces were the same as those of a previously reported patient with homozygous CDK9 variants. The ocular phenotype included blepharophimosis, lacrimal duct obstruction, eyelid dermoids, Duane syndrome-like abduction deficit, and congenital cataracts. Optical coherence tomography and electroretinography evaluations revealed severe retinal dystrophy had developed at an early age. Trio-based whole-exome sequencing identified compound heterozygous variants in CDK9 [p.(A288T) of maternal origin and p.(R303C) of paternal origin] in the patient. Variants' kinase activities were reduced compared with wild type. We concluded that CDK9 biallelic variants cause a CHARGE-like malformation syndrome with retinal dystrophy as a distinguishing feature.
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Blefarofimosis/genética , Síndrome CHARGE/genética , Quinasa 9 Dependiente de la Ciclina/genética , Distrofias Retinianas/genética , Alelos , Blefarofimosis/diagnóstico , Blefarofimosis/patología , Síndrome CHARGE/diagnóstico , Síndrome CHARGE/diagnóstico por imagen , Síndrome CHARGE/patología , Niño , Labio Leporino/diagnóstico por imagen , Labio Leporino/genética , Labio Leporino/patología , Fisura del Paladar/diagnóstico por imagen , Fisura del Paladar/genética , Fisura del Paladar/patología , Electrorretinografía , Homocigoto , Humanos , Obstrucción del Conducto Lagrimal/diagnóstico , Obstrucción del Conducto Lagrimal/genética , Obstrucción del Conducto Lagrimal/patología , Masculino , Mutación/genética , Linaje , Fenotipo , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/diagnóstico por imagen , Distrofias Retinianas/patología , Tomografía de Coherencia Óptica , Secuenciación del ExomaRESUMEN
Developmental and epileptic encephalopathy (DEE) represents a group of neurodevelopmental disorders characterized by infantile-onset intractable seizures and unfavorable prognosis of psychomotor development. To date, hundreds of genes have been linked to the onset of DEE. GNAO1 is a DEE-associated gene encoding the alpha-O1 subunit of guanine nucleotide-binding protein (GαO ). Despite the increasing number of reported children with GNAO1 encephalopathy, the molecular mechanisms underlying their neurodevelopmental phenotypes remain elusive. We herein present that co-immunoprecipitation and mass spectrometry analyses identified another DEE-associated protein, SPTAN1, as an interacting partner of GαO . Silencing of endogenous Gnao1 attenuated the neurite outgrowth and calcium-dependent signaling. Inactivation of GNAO1 in human-induced pluripotent stem cells gave rise to anomalous brain organoids that only weakly expressed SPTAN1 and Ankyrin-G. Furthermore, GNAO1-deficient organoids failed to conduct synchronized firing to adjacent neurons. These data indicate that GαO and other DEE-associated proteins organize the cytoskeletal remodeling and functional polarity of neurons in the developing brain.
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Citoesqueleto/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Animales , Encéfalo/metabolismo , Encefalopatías/metabolismo , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Trastornos del Neurodesarrollo/metabolismo , Neuronas/metabolismo , FenotipoRESUMEN
Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.
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Nucleótidos/metabolismo , Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Embrión no Mamífero/anomalías , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Larva/genética , Larva/fisiología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/patología , Proteína Básica de Mielina/genética , Nucleótidos/genética , Sirolimus/farmacología , Pez Cebra/embriologíaRESUMEN
Fragments of transfer RNA (tRNA), derived either from pre-tRNA or mature tRNA, have been discovered to play an essential role in the pathogenesis of various disorders such as neurodegenerative disease. CLP1 is an RNA kinase involved in tRNA biogenesis, and mutations in its encoding gene are responsible for pontocerebellar hypoplasia type-10. Mutation of the CLP1 gene results in the accumulation of tRNA fragments of several different kinds. These tRNA fragments are expected to be associated with the disease pathogenesis. However, it is still unclear which of the tRNA fragments arising from the CLP1 gene mutation has the greatest impact on the onset of neuronal disease. We found that 5' tRNA fragments derived from tyrosine pre-tRNA (5' Tyr-tRF) caused p53-dependent neuronal cell death predominantly more than other types of tRNA fragment. We also showed that 5' Tyr-tRF bound directly to pyruvate kinase M2 (PKM2). Injection of zebrafish embryos with PKM2 mRNA ameliorated the neuronal defects induced in zebrafish embryos by 5' Tyr-tRF. Our findings partially uncovered a mechanistic link between 5' Tyr-tRF and neuronal cell death that is regulated by PKM2.
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Neuronas/enzimología , Neuronas/patología , Piruvato Quinasa/metabolismo , Precursores del ARN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Tirosina/metabolismo , Animales , Muerte Celular , Diferenciación Celular , Línea Celular , Embrión no Mamífero/metabolismo , Humanos , Pez Cebra/embriologíaRESUMEN
Cerebellar Purkinje cells (PCs) are the sole output neurons of the cerebellar cortex, and damage to PCs results in motor deficits. Spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease), a hereditary neurodegenerative disease, is caused by an abnormal expansion of the polyglutamine tract in the causative ATXN3 protein. SCA3 affects a wide range of cells in the central nervous system, including those in the cerebellum. To unravel SCA3 pathology, we used adeno-associated virus serotype 9 (AAV9) vectors to express full-length ATXN3 with an abnormally expanded 89 polyglutamine stretch (ATXN3[Q89]) in cerebellar neurons of mature wild-type mice. Mice expressing ATXN3[Q89] exhibited motor impairment in a manner dependent on the viral titer. Immunohistochemistry of the cerebellum showed ubiquitinated nuclear aggregates in PCs; degeneration of PC dendrites; and a significant decrease in multiple proteins including retinoid-related orphan receptor α (RORα), a transcription factor, and type 1 metabotropic glutamate receptor (mGluR1) signaling molecules. Patch clamp analysis of ATXN3[Q89]-expressing PCs revealed marked defects in mGluR1 signaling. Notably, the emergence of behavioral, morphological, and functional defects was inhibited by a single injection of SR1078, an RORα/γ agonist. These results suggest that RORα plays a key role in mutant ATXN3-mediated aberrant phenotypes and that the pharmacological enhancement of RORα could function as a method for therapeutic intervention in SCA3.
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Ataxina-3/metabolismo , Enfermedad de Machado-Joseph/metabolismo , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Células de Purkinje/metabolismo , Proteínas Represoras/metabolismo , Animales , Ataxina-3/genética , Dendritas/patología , Humanos , Enfermedad de Machado-Joseph/patología , Ratones Endogámicos C57BL , Péptidos/genética , Agregación Patológica de Proteínas/metabolismo , Células de Purkinje/patología , Receptores de Glutamato Metabotrópico/metabolismo , Proteínas Represoras/genética , Transducción de SeñalRESUMEN
Zebrafish embryos are useful to study haematopoietic gene function in vertebrates, although lack of antibodies to zebrafish proteins has limited the purification of specific cell populations. Here, we purified primitive zebrafish erythrocytes using 1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione (DRAQ5TM ), a DNA-staining fluorescent dye. At 48-h post-fertilization, we sorted small-sized cells from embryos using forward scatter and found that they consisted of DRAQ5high and DRAQ5low populations. DRAQ5high cells contained haemoglobin, lacked myeloperoxidase activity and expressed high levels of embryonic globin (hbae3 and hbbe1.1) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5TM analysis of gata1:dsRed transgenic embryos, we purified primitive DRAQ5high dsRed+ erythrocytes from haematopoietic progenitor cells. Using this method, we identified docking protein 2 (Dok2) as functioning in differentiation of primitive erythrocytes. We conclude that DRAQ5TM -based flow cytometry enables purification of primitive zebrafish erythrocytes.
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Eritrocitos/citología , Eritrocitos/metabolismo , Hematopoyesis , Animales , Biomarcadores , Separación Celular/métodos , Citometría de Flujo , Regulación de la Expresión Génica , Hematopoyesis/genética , Inmunofenotipificación , Especificidad de Órganos/genética , Pez CebraRESUMEN
Methylation of histone H3 Lys 9 and Lys 27 (H3K9 and H3K27) is associated with transcriptional silencing. Here we show that KDM7, a JmjC domain-containing protein, catalyzes demethylation of both mono- or dimethylated H3K9 and H3K27. Inhibition of KDM7 orthologs in zebrafish resulted in developmental brain defects. KDM7 interacts with the follistatin gene locus, and KDM7 depletion in mammalian neuronal cells suppressed follistatin gene transcription in association with increased levels of dimethylated H3K9 and H3K27. Our findings identify KDM7 as a dual demethylase for H3K9 and H3K27 that functions as an eraser of silencing marks on chromatin during brain development.
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Encéfalo/embriología , Encéfalo/enzimología , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Células Cultivadas , Embrión no Mamífero/embriología , Embrión no Mamífero/enzimología , Folistatina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Neuronas/metabolismo , Pez Cebra/embriologíaRESUMEN
Mice null for wild-type p53-induced phosphatase 1 (WIP1) display defects in testis development and spermatogenesis, resulting in reduced fertility. However, the molecular mechanism underlying these abnormalities in the testis remains uncharacterized. We report that the phosphatase activity of WIP1 increases Wnt activity through Nemo-like kinase (NLK). WIP1 directly interacted with NLK, which is highly homologous to p38 MAPK, a WIP1 substrate, and dephosphorylated its activation site. The WIP1-mediated inhibition of NLK activity markedly decreased the phosphorylation of lymphoid enhancer-binding factor 1 (LEF1), enhancing its interaction with ß-catenin. Additionally, WIP1 depletion impaired germ cell development, as evidenced by the expression of Oct4 and the germ cell-specific markers Ddx4, Nanos3 and Dnd1 during the development of germ cells from Oct4-GFP transgenic (OG2) mouse embryonic stem cells (mESCs). The expression of WIP1, whose level was significantly lower after the differentiation of germ cells from mESCs, occurred in parallel with the expression of germ cell development markers and SRY-box 17 (Sox17), a downstream target of Wnt. These results indicate that WIP1 is essential for germ cell development, which is known to require Wnt activity.
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Células Germinativas/citología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células Madre Embrionarias de Ratones/citología , Proteína Fosfatasa 2C/metabolismo , Proteínas Wnt/metabolismo , Animales , Diferenciación Celular , Línea Celular , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Células Germinativas/metabolismo , Células HEK293 , Humanos , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Masculino , Ratones , Ratones Transgénicos , Células Madre Embrionarias de Ratones/metabolismo , Fosforilación , Proteína Fosfatasa 2C/genética , Proteínas Serina-Treonina Quinasas , Testículo/citología , Testículo/metabolismo , beta Catenina/metabolismoRESUMEN
Nemo-like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/ß-catenin signalling. However, the roles of NLK in Wnt/ß-catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor-1 (Lef1)-mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC-like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/ß-catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/ß-catenin signalling pathway.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Células Madre/fisiología , Factores de Transcripción/metabolismo , Proteína Wnt1/metabolismo , Proteínas de Pez Cebra/metabolismo , beta Catenina/metabolismo , Animales , Encéfalo/embriología , Encéfalo/fisiología , Células Cultivadas , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Fosforilación , Pez CebraRESUMEN
DP1, a dimerization partner protein of the transcription factor E2F, is known to inhibit Wnt/ß-catenin signalling along with E2F, although the function of DP1 itself was not well characterized. Here, we present a novel dual regulatory mechanism of Wnt/ß-catenin signalling by DP1 independent from E2F. DP1 negatively regulates Wnt/ß-catenin signalling by inhibiting Dvl-Axin interaction and by enhancing poly-ubiquitination of ß-catenin. In contrast, DP1 positively modulates the signalling upon Wnt stimulation, via increasing cytosolic ß-catenin and antagonizing the kinase activity of NLK. In Xenopus embryos, DP1 exerts both positive and negative roles in Wnt/ß-catenin signalling during anteroposterior neural patterning. From subcellular localization analyses, we suggest that the dual roles of DP1 in Wnt/ß-catenin signalling are endowed by differential nucleocytoplasmic localizations. We propose that these dual functions of DP1 can promote and stabilize biphasic Wnt-on and Wnt-off states in response to a gradual gradient of Wnt/ß-catenin signalling to determine differential cell fates.
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Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción DP1/metabolismo , Proteínas Wnt/metabolismo , Xenopus/embriología , Animales , Transducción de Señal , beta Catenina/metabolismoRESUMEN
Sonic hedgehog (Shh) is a secreted morphogen that controls the patterning and growth of various tissues in the developing vertebrate embryo, including the central nervous system. Ablation of the FK506-binding protein 38 (FKBP38) gene results in activation of the Shh signaling pathway in mouse embryos, but the molecular mechanism by which FKBP38 suppresses Shh signaling has remained unclear. With the use of a proteomics approach, we have now identified ANKMY2, a protein with three ankyrin repeats and a MYND (myeloid, Nervy, and DEAF-1)-type Zn(2+) finger domain, as a molecule that interacts with FKBP38. Co-immunoprecipitation analysis confirmed that endogenous FKBP38 and ANKMY2 interact in the mouse brain. Depletion or overexpression of ANKMY2 resulted in down- and up-regulation of Shh signaling, respectively, in mouse embryonic fibroblasts. Furthermore, combined depletion of both FKBP38 and ANKMY2 attenuated Shh signaling in these cells, suggesting that ANKMY2 acts downstream of FKBP38 to activate the Shh signaling pathway. Targeting of the zebrafish ortholog of mouse Ankmy2 (ankmy2a) in fish embryos with an antisense morpholino oligonucleotide conferred a phenotype reflecting loss of function of the Shh pathway, suggesting that the regulation of Shh signaling by ANKMY2 is conserved between mammals and fish. Our findings thus indicate that the FKBP38-ANKMY2 axis plays a key role in regulation of Shh signaling in vivo.
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Proteínas Portadoras/genética , Proteínas Hedgehog/metabolismo , Transducción de Señal/genética , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Ratones , Proteínas de Unión a Tacrolimus/genética , Transactivadores/metabolismo , Pez Cebra/genéticaRESUMEN
Hepatocellular carcinoma (HCC), the third most common cause of cancer-related deaths worldwide, lacks effective medical therapy. Large subsets of HCC demonstrate Wnt/ß-catenin activation, making this an attractive therapeutic target. We report strategy and characterization of a novel small-molecule inhibitor, ICG-001, known to affect Wnt signaling by disrupting ß-catenin-CREB binding protein interactions. We queried the ZINC online database for structural similarity to ICG-001 and identified PMED-1 as the lead compound, with ≥70% similarity to ICG-001. PMED-1 significantly reduced ß-catenin activity in hepatoblastoma and several HCC cells, as determined by TOPflash reporter assay, with an IC50 ranging from 4.87 to 32 µmol/L. Although no toxicity was observed in primary human hepatocytes, PMED-1 inhibited Wnt target expression in HCC cells, including those with CTNNB1 mutations, and impaired cell proliferation and viability. PMED-1 treatment decreased ß-catenin-CREB binding protein interactions without affecting total ß-catenin levels or activity of other common kinases. PMED-1 treatment of Tg(OTM:d2EGFP) zebrafish expressing GFP under the ß-catenin/Tcf reporter led to a notable decrease in ß-catenin activity. The PMED effect on ß-catenin signaling lasted from 12 to 24 hours in vitro and 6 to 15 hours in vivo. Thus, using a rapid and cost-effective computational methodology, we have identified a novel and specific small-molecule inhibitor of Wnt signaling that may have implications for HCC treatment.