RESUMEN
Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.
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Antioxidantes , Benzotiazoles , Modelos Animales de Enfermedad , Disostosis Mandibulofacial , Estrés Oxidativo , Especies Reactivas de Oxígeno , Tolueno/análogos & derivados , Proteína p53 Supresora de Tumor , Proteínas de Pez Cebra , Pez Cebra , Animales , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Disostosis Mandibulofacial/genética , Disostosis Mandibulofacial/tratamiento farmacológico , Antioxidantes/farmacología , Benzotiazoles/farmacología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Tolueno/farmacología , Cresta Neural/efectos de los fármacos , Cresta Neural/metabolismo , Apoptosis/efectos de los fármacos , ARN Polimerasa I/antagonistas & inhibidores , ARN Polimerasa I/metabolismo , ARN Polimerasa I/genéticaRESUMEN
The peptidyl prolyl cis-trans isomerase Pin1 plays vital roles in diverse cellular processes and pathological conditions. NeuroD is a differentiation and survival factor for a subset of neurons and pancreatic endocrine cells. Although multiple phosphorylation events are known to be crucial for NeuroD function, their mechanisms remain elusive. In this study, we demonstrate that zebrafish embryos deficient in Pin1 displayed phenotypes resembling those associated with NeuroD depletion, characterized by defects in formation of mechanosensory hair cells. Furthermore, zebrafish Pin1 interacts with NeuroD in a phosphorylation-dependent manner. In Pin1-deficient cell lines, NeuroD is rapidly degraded. However, the protein stability of NeuroD is restored upon overexpression of Pin1. These findings suggest that Pin1 functionally regulates NeuroD protein levels by post-phosphorylation cis-trans isomerization during neuronal specification.
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Bone has multiple functions in animals, such as supporting the body for mobility. The zebrafish skeleton is composed of craniofacial and axial skeletons. It shares a physiological curvature and consists of a similar number of vertebrae as humans. Bone degeneration and malformations have been widely studied in zebrafish as human disease models. High-resolution imaging and different bone properties such as density and volume can be obtained using micro-computed tomography (micro-CT). This study aimed to understand the possible changes in the structure and bone mineral density (BMD) of the vertebrae and craniofacial skeleton with age (4, 12 and 24 months post fertilisation [mpf]) in zebrafish. Our data showed that the BMD in the vertebrae and specific craniofacial skeleton (mandibular arch, ceratohyal and ethmoid plate) of 12 and 24 mpf fish were higher than that of the 4 mpf fish. In addition, we found the age-dependent increase in BMD was not ubiquitously observed in facial bones, and such differences were not correlated with bone type. In summary, such additional information on the craniofacial skeleton could help in understanding bone development throughout the lifespan of zebrafish.
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Densidad Ósea , Pez Cebra , Animales , Humanos , Microtomografía por Rayos X/métodos , Huesos Faciales/diagnóstico por imagen , Columna VertebralRESUMEN
BACKGROUND: Restless legs syndrome is a highly prevalent comorbidity of migraine; however, its genetic contributions remain unclear. OBJECTIVES: To identify the genetic variants of restless legs syndrome in migraineurs and to investigate their potential pathogenic roles. METHODS: We conducted a two-stage genome-wide association study (GWAS) to identify susceptible genes for restless legs syndrome in 1,647 patients with migraine, including 264 with and 1,383 without restless legs syndrome, and also validated the association of lead variants in normal controls unaffected with restless legs syndrome (n = 1,053). We used morpholino translational knockdown (morphants), CRISPR/dCas9 transcriptional knockdown, transient CRISPR/Cas9 knockout (crispants) and gene rescue in one-cell stage embryos of zebrafish to study the function of the identified genes. RESULTS: We identified two novel susceptibility loci rs6021854 (in VSTM2L) and rs79823654 (in CCDC141) to be associated with restless legs syndrome in migraineurs, which remained significant when compared to normal controls. Two different morpholinos targeting vstm2l and ccdc141 in zebrafish demonstrated behavioural and cytochemical phenotypes relevant to restless legs syndrome, including hyperkinetic movements of pectoral fins and decreased number in dopaminergic amacrine cells. These phenotypes could be partially reversed with gene rescue, suggesting the specificity of translational knockdown. Transcriptional CRISPR/dCas9 knockdown and transient CRISPR/Cas9 knockout of vstm2l and ccdc141 replicated the findings observed in translationally knocked-down morphants. CONCLUSIONS: Our GWAS and functional analysis suggest VSTM2L and CCDC141 are highly relevant to the pathogenesis of restless legs syndrome in migraineurs.
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Síndrome de las Piernas Inquietas , Animales , Estudio de Asociación del Genoma Completo , Humanos , Síndrome de las Piernas Inquietas/complicaciones , Síndrome de las Piernas Inquietas/genética , Pez Cebra/genéticaRESUMEN
Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.
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Desarrollo Embrionario/genética , Proteínas de Homeodominio/genética , Proteína Jagged-1/genética , Proteína Jagged-2/genética , Proteínas del Tejido Nervioso/genética , Receptor Notch1/genética , Proteínas de Pez Cebra/genética , Animales , Proteínas de Unión al Calcio/genética , Diferenciación Celular/genética , Ectodermo/crecimiento & desarrollo , Epidermis/crecimiento & desarrollo , Queratinocitos/citología , Queratinocitos/metabolismo , Moco/metabolismo , Proteínas Mutantes/genética , Receptores Notch/genética , Transducción de Señal/genética , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Mutations of the Gon4l/udu gene in different organisms give rise to diverse phenotypes. Although the effects of Gon4l/Udu in transcriptional regulation have been demonstrated, they cannot solely explain the observed characteristics among species. To further understand the function of Gon4l/Udu, we used yeast two-hybrid (Y2H) screening to identify interacting proteins in zebrafish and mouse systems, confirmed the interactions by co-immunoprecipitation assay, and found four novel Gon4l-interacting proteins: BRCA1 associated protein-1 (Bap1), DNA methyltransferase 1 (Dnmt1), Tho complex 1 (Thoc1, also known as Tho1 or HPR1), and Cryptochrome circadian regulator 3a (Cry3a). Furthermore, all known Gon4l/Udu-interacting proteins-as found in this study, in previous reports, and in online resources-were investigated by Phenotype Enrichment Analysis. The most enriched phenotypes identified include increased embryonic tissue cell apoptosis, embryonic lethality, increased T cell derived lymphoma incidence, decreased cell proliferation, chromosome instability, and abnormal dopamine level, characteristics that largely resemble those observed in reported Gon4l/udu mutant animals. Similar to the expression pattern of udu, those of bap1, dnmt1, thoc1, and cry3a are also found in the brain region and other tissues. Thus, these findings indicate novel mechanisms of Gon4l/Udu in regulating CpG methylation, histone expression/modification, DNA repair/genomic stability, and RNA binding/processing/export.
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Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Línea Celular , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Ratones , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina Tiolesterasa/metabolismo , Pez CebraRESUMEN
Skin depigmentation diseases, such as vitiligo, are pigmentation disorders that often destroy melanocytes. However, their pathological mechanisms remain unclear, and therefore, promising treatments or prevention has been lacking. Here, we demonstrate that a zebrafish insertional mutant showing a significant reduction of nicastrin transcript possesses melanosome maturation defect, Tyrosinase-dependent mitochondrial swelling, and melanophore cell death. The depigmentation phenotypes are proven to be a result of γ-secretase inactivation. Furthermore, live imaging demonstrates that macrophages are recruited to and can phagocytose melanophore debris. Thus, we characterize a potential zebrafish depigmentation disease model, a nicastrinhi1384 mutant, which can be used for further treatment or drug development of diseases related to skin depigmentation and/or inflammation.
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Secretasas de la Proteína Precursora del Amiloide/genética , Hipopigmentación/genética , Glicoproteínas de Membrana/genética , Monofenol Monooxigenasa/metabolismo , Piel/inmunología , Proteínas de Pez Cebra/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Embrión no Mamífero , Humanos , Hipopigmentación/inmunología , Hipopigmentación/patología , Melanosomas/inmunología , Melanosomas/metabolismo , Melanosomas/ultraestructura , Glicoproteínas de Membrana/metabolismo , Microscopía Electrónica de Transmisión , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/genética , Mutación , Piel/patología , Pigmentación de la Piel/efectos de los fármacos , Pigmentación de la Piel/genética , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Interkinetic nuclear migration (INM) is a process by which nuclei oscillate between the basal and apical surfaces of epithelial cells in coordination with the cell cycle. The cytoskeletal machinery including microtubules and actin has been reported to drive apical INM; however, the role of nuclear proteins in this process has yet to be fully elucidated. Here, we investigated the function of a SUN-domain protein, Sun1, in zebrafish. We found that zebrafish sun1 is highly expressed in the ventricular zone of the brain. Knocking down sun1 with antisense morpholino oligonucleotides reduced the abundance of nestin- and gfap-expressing neural stem cells and progenitor cells. The live-cell imaging results showed that sun1 morphant cells migrated toward the basal side during the S phase but failed to migrate apically during the G2 phase. On the contrary, the passive stochastic movement during the G2 phase was unaffected. Furthermore, down regulation of sun1 was shown to reduce the expression of genes associated with the Notch pathway, whereas the expression of genes in the Wnt pathway was less perturbed. Findings from this research suggest that the Sun1-mediated nucleo-cytoskeletal interaction contributes to apical nuclear migration, and may thus affect exposure to Notch signal, thereby altering the composition of the progenitor pool in the embryonic neurogenesis of zebrafish.
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Núcleo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neurogénesis/fisiología , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Pez Cebra/metabolismo , Actinas/metabolismo , Animales , Ciclo Celular/fisiología , División Celular/fisiología , Proliferación Celular/fisiología , Centrosoma/metabolismo , Citoesqueleto/metabolismo , Células-Madre Neurales , Neuronas/metabolismoRESUMEN
Treacher Collins syndrome (TCS) is a rare congenital birth disorder (1 in 50,000 live births) characterized by severe craniofacial defects. Recently, the authors' group unfolded the pathogenesis of polr1c Type 3 TCS by using the zebrafish model. Facial development depends on the neural crest cells, in which polr1c plays a role in regulating their expression. In this study, the authors aimed to identify the functional time window of polr1c in TCS by the use of photo-morpholino to restore the polr1c expression at different time points. Results suggested that the restoration of polr1c at 8 hours after fertilization could rescue the TCS facial malformation phenotype by correcting the neural crest cell expression, reducing the cell death, and normalizing the p53 mRNA expression level in the rescued morphants. However, such recovery could not be reproduced if the polr1c is restored after 30 hours after fertilization.
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ARN Polimerasas Dirigidas por ADN/fisiología , Terapias Fetales/métodos , Terapia Genética/métodos , Disostosis Mandibulofacial/prevención & control , Animales , Muerte Celular/genética , ARN Polimerasas Dirigidas por ADN/genética , Modelos Animales de Enfermedad , Desarrollo Embrionario/fisiología , Cara/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes p53 , Disostosis Mandibulofacial/embriología , Disostosis Mandibulofacial/genética , Disostosis Mandibulofacial/patología , Morfolinos , Cresta Neural/metabolismo , Fenotipo , Factores de Tiempo , Pez CebraRESUMEN
mibnn2002, identified from an allele screen, shows early segmentation defect and severe cell death phenotypes, which are different from those of other described mib mutant alleles. We have previously reported its defects in somitogenesis and identified its origin of mutation, a large deletion in LG2. The report here is a continuous study, where we applied the bioinformatics analysis to profile the genetic background of mibnn2002 mutants. By comparing the transcriptomic data of mibnn2002 mutants with those of AB wild-type, a total of 1945 differentially expressed genes were identified, including 685 up- and 1260 down-regulated genes. The Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis and Ingenuity Pathway Analysis (IPA) identified the enriched pathways and their related biological functions. Our data further demonstrated that the defects in the somitogenesis were related to the down-regulated segmentation genes, such as foxc1a, smyhc1, myod1 and mylpfa.
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Perfilación de la Expresión Génica/métodos , Eliminación de Secuencia , Somitos/anomalías , Ubiquitina-Proteína Ligasas/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Biología Computacional/métodos , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Pez Cebra/genéticaRESUMEN
Nasopharyngeal carcinoma (NPC) is a serious health problem in China and Southeast Asia. Relapse is the major cause of mortality, but mechanisms of relapse are mysterious. Epstein-Barr virus (EBV) reactivation and host genomic instability (GI) have correlated with NPC development. Previously, we reported that lytic early genes DNase and BALF3 induce genetic alterations and progressive malignancy in NPC cells, implying lytic proteins may be required for NPC relapse. In this study, we show that immediate early gene BRLF1 induces chromosome mis-segregation and genomic instability in the NPC cells. Similar phenomenon was also demonstrated in 293 and zebrafish embryonic cells. BRLF1 nuclear localization signal (NLS) mutant still induced genomic instability and inhibitor experiments revealed that BRLF1 interferes with chromosome segregation and induces genomic instability by activating Erk signaling. Furthermore, the chromosome aberrations and tumorigenic features of NPC cells were significantly increased with the rounds of BRLF1 expression, and these cells developed into larger tumor nodules in mice. Therefore, BRLF1 may be the important factor contributing to NPC relapse and targeting BRLF1 may benefit patients.
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Although the zebrafish interrenal tissue has been used as a model for steroidogenesis and genesis of the adrenal gland, its specification and morphogenesis remains largely unclear. In the present study, we explored how the Wilms tumor 1 (WT1)-expressing cells are segregated from the SF-1-expressing steroidogenic cells in the zebrafish model. The interrenal tissue precursors expressing ff1b, the equivalent of mammalian SF-1, were derived from wt1-expressing pronephric primordia in the zebrafish embryo. Through histochemistry and in situ hybridization, we demonstrated that the size of functionally differentiated interrenal tissue was substantially increased on global inhibition of the Notch signaling pathway and was accompanied by a disrupted segregation between the wt1- and ff1b-expressing cells. As the Notch pathway was conditionally activated during interrenal specification, differentiation, but not ff1b expression, of interrenal tissue was drastically compromised. In embryos deficient for Notch ligands jagged 1b and 2b, transgenic reporter activity of wt1b promoter was detected within the steroidogenic interrenal tissue. In conclusion, our results indicate that Jagged-Notch signaling is required (1) for segregation between wt1-expressing cells and differentiated steroidogenic tissue; and (2) to modulate the extent of functional differentiation in the steroidogenic interrenal tissue.
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Proteína Jagged-1/genética , Proteína Jagged-2/genética , Receptores Notch/genética , Transducción de Señal/genética , Proteínas WT1/genética , Proteínas de Pez Cebra/genética , Animales , Animales Modificados Genéticamente , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Riñón Cefálico/citología , Riñón Cefálico/embriología , Riñón Cefálico/metabolismo , Hibridación in Situ , Glándula Interrenal/citología , Glándula Interrenal/embriología , Glándula Interrenal/metabolismo , Proteína Jagged-1/metabolismo , Proteína Jagged-2/metabolismo , Receptores Notch/metabolismo , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Esteroides/biosíntesis , Proteínas WT1/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
High-throughput screening with a loss-of-function strategy is a logical and efficient way to identify novel genes involved in biological processes of interest. In zebrafish, morpholinos have been developed as a convenient tool to knock down gene expression. Here, we describe procedures for systematic screening using morpholinos in zebrafish to identify novel deubiquitylases involved in convergent extension during gastrulation. In this example, we examine candidates based on embryonic morphology and molecular signals of whole mount in situ hybridization assay.
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Regulación del Desarrollo de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento/métodos , Hibridación in Situ/métodos , Morfolinos/genética , Pez Cebra/embriología , Pez Cebra/genética , Animales , Embrión no Mamífero , Gastrulación/genética , Técnicas de Silenciamiento del Gen/métodos , Microinyecciones/métodos , Morfolinos/farmacología , Proteasas Ubiquitina-Específicas/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In the past three decades, the number of zebrafish laboratories has significantly increased in Taiwan. The Taiwan Zebrafish Core Facility (TZCF), a government-funded core facility, was launched to serve this growing community. The Core Facility was built on two sites, one located at the National Health Research Institutes (NHRI, called Taiwan Zebrafish Core Facility at NHRI or TZeNH) and the other is located at the Academia Sinica (Taiwan Zebrafish Core Facility at AS a.k.a. TZCAS). The total surface area of the TZCF is about 180 m(2) encompassing 2880 fish tanks. Each site has a separate quarantine room and centralized water recirculating systems, monitoring key water parameters. To prevent diseases, three main strategies have been implemented: (1) imported fish must be quarantined; (2) only bleached embryos are introduced into the main facilities; and (3) working practices were implemented to minimize pathogen transfer between stocks and facilities. Currently, there is no health program in place; however, a fourth measure for the health program, specific regular pathogen tests, is being planned. In March 2015, the TZCF at NHRI has been AAALAC accredited. It is our goal to ensure that we provide "disease-free" fish and embryos to the Taiwanese research community.
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Crianza de Animales Domésticos/métodos , Animales de Laboratorio , Acuicultura/métodos , Pez Cebra , Crianza de Animales Domésticos/instrumentación , Crianza de Animales Domésticos/organización & administración , Animales , Acuicultura/instrumentación , Acuicultura/organización & administración , Modelos Animales , TaiwánRESUMEN
Treacher Collins Syndrome (TCS) is a rare congenital birth disorder (1 in 50,000 live births) characterized by severe craniofacial defects, including the downward slanting palpebral fissures, hypoplasia of the facial bones, and cleft palate (CP). Over 90% of patients with TCS have a mutation in the TCOF1 gene. However, some patients exhibit mutations in two new causative genes, POLR1C and POLR1D, which encode subunits of RNA polymerases I and III, that affect ribosome biogenesis. In this study, we examine the role of POLR1C in TCS using zebrafish as a model system. Our data confirmed that polr1c is highly expressed in the facial region, and dysfunction of this gene by knockdown or knock-out resulted in mis-expression of neural crest cells during early development that leads to TCS phenotype. Next generation sequencing and bioinformatics analysis of the polr1c mutants further demonstrated the up-regulated p53 pathway and predicted skeletal disorders. Lastly, we partially rescued the TCS facial phenotype in the background of p53 mutants, which supported the hypothesis that POLR1C-dependent type 3 TCS is associated with the p53 pathway.
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Disostosis Mandibulofacial/genética , Disostosis Mandibulofacial/patología , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Mutación , Cresta Neural/metabolismo , Cresta Neural/patología , Proteína p53 Supresora de Tumor/genética , Pez Cebra/embriologíaRESUMEN
Familial exudative vitreoretinopathy (FEVR) belongs to a group of genetically and clinically heterogeneous disorders in retinal vascular development. To date, in approximately 50% of patients with FEVR, pathogenic mutations have been detected in FZD4, LRP5, TSPAN12, NDP and ZNF408. In this study, we identified two heterozygous frameshift mutations in RCBTB1 from three Taiwanese cases through exome sequencing. In patient-derived lymphoblastoid cell lines (LCLs), the protein level of RCBTB1 is approximately half that of unaffected control LCLs, which is indicative of a haploinsufficiency mechanism. By employing transient transfection and reporter assays for the transcriptional activity of ß-catenin, we demonstrated that RCBTB1 participates in the Norrin/FZD4 signaling pathway and that knockdown of RCBTB1 by shRNA significantly reduced nuclear accumulation of ß-catenin under Norrin and Wnt3a treatments. Furthermore, transgenic fli1:EGFP zebrafish with rcbtb1 knockdown exhibited anomalies in intersegmental and intraocular vessels. These results strongly support that reduced RCBTB1 expression may lead to defects in angiogenesis through the Norrin-dependent Wnt pathway, and that RCBTB1 is a putative genetic cause of vitreoretinopathies.
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Factores de Intercambio de Guanina Nucleótido/genética , Haploinsuficiencia , Neovascularización Fisiológica , Enfermedades de la Retina/genética , Telangiectasia Retiniana/genética , Análisis de Secuencia de ADN/métodos , Línea Celular , Exoma , Enfermedades Hereditarias del Ojo , Proteínas del Ojo/metabolismo , Vitreorretinopatías Exudativas Familiares , Femenino , Mutación del Sistema de Lectura , Predisposición Genética a la Enfermedad , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Masculino , Proteínas del Tejido Nervioso/metabolismo , Taiwán , Vía de Señalización WntRESUMEN
BACKGROUND: Notch signaling has been conserved throughout evolution and plays a fundamental role in various neural developmental processes and the pathogenesis of several human cancers and genetic disorders. However, how Notch signaling regulates various cellular processes remains unclear. Although Deltex proteins have been identified as cytoplasmic downstream elements of the Notch signaling pathway, few studies have been reported on their physiological role. RESULTS: We isolated zebrafish deltex1 (dtx1) and showed that this gene is primarily transcribed in the developing nervous system, and its spatiotemporal expression pattern suggests a role in neural differentiation. The transcription of dtx1 was suppressed by the direct binding of the Notch downstream transcription factors Her2 and Her8a. Overexpressing the complete coding sequence of Dtx1 was necessary for inducing neuronal and glial differentiation. By contrast, disrupting Dtx1 expression by using a Dtx1 construct without the RING finger domain reduced neuronal and glial differentiation. This effect was phenocopied by the knockdown of endogenous Dtx1 expression by using morpholinos, demonstrating the essential function of the RING finger domain and confirming the knockdown specificity. Cell proliferation and apoptosis were unaltered in Dtx1-overexpressed and -deficient zebrafish embryos. Examination of the expression of her2 and her8a in embryos with altered Dtx1 expression showed that Dxt1-induced neuronal differentiation did not require a regulatory effect on the Notch-Hairy/E(Spl) pathway. However, both Dtx1 and Notch activation induced glial differentiation, and Dtx1 and Notch activation negatively inhibited each other in a reciprocal manner, which achieves a proper balance for the expression of Dtx1 and Notch to facilitate glial differentiation. We further confirmed that the Dtx1-Notch-Hairy/E(Spl) cascade was sufficient to induce neuronal and glial differentiation by concomitant injection of an active form of Notch with dtx1, which rescued the neuronogenic and gliogenic defects caused by the activation of Notch signaling. CONCLUSIONS: Our results demonstrated that Dtx1 is regulated by Notch-Hairy/E(Spl) signaling and is a major factor specifically regulating neural differentiation. Thus, our results provide new insights into the mediation of neural development by the Notch signaling pathway.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Receptores Notch/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Neurogénesis/fisiología , Neuroglía/citología , Neuronas/citología , Receptores Notch/genética , Transducción de Señal/fisiología , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
mib(nn2002), found from an allele screen, showed early segmentation defect and severe cell death phenotypes, which are different from previously known mib mutants. Despite distinct morphological phenotypes, the typical mib molecular phenotypes: her4 down-regulation, neurogenic phenotype and cold sensitive dlc expression pattern, still remained. The linkage analysis also indicated that mib(nn2002) is a new mib allele. Failure of specification in anterior 7-10 somites is likely due to lack of foxc1a expression in mib(nn2002) homozygotes. Somites and somite markers gradually appeared after 7-10 somite stage, suggesting that foxc1a is only essential for the formation of anterior 7-10 somites. Apoptosis began around 16-somite stage with p53 up-regulation. To find the possible links of mib, foxc1a and apoptosis, transcriptome analysis was employed. About 140 genes, including wnt3a, foxc1a and mib, were not detected in the homozygotes. Overexpression of foxc1a mRNA in mib(nn2002) homozygotes partially rescued the anterior somite specification. In the process of characterizing mib(nn2002) mutation, we integrated the scaffolds containing mib locus into chromosome 2 (or linkage group 2, LG2) based on synteny comparison and transcriptome results. Genomic PCR analysis further supported the conclusion and showed that mib(nn2002) has a chromosomal deletion with the size of about 9.6 Mbp.