RESUMEN
North Carolina macular dystrophy (NCMD) is a rare autosomal-dominant disease affecting macular development. The disease is caused by non-coding single-nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1. To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 promoters and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays. Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two NCMD-associated non-coding SNVs that we identified herein. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, suggesting that this region is a PRDM13 enhancer. Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis. Overall, this study provides insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy.
Asunto(s)
Distrofias Hereditarias de la Córnea , Tomografía de Coherencia Óptica , Adulto , Animales , Humanos , Linaje , Retina/metabolismo , Xenopus laevis/genéticaRESUMEN
In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.
RESUMEN
The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1-3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Extremidades/embriología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Deformidades Congénitas de las Extremidades/genética , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Fibroblastos , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Masculino , Ratones , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/metabolismo , Fenotipo , Receptores Acoplados a Proteínas G/deficiencia , Ubiquitina-Proteína Ligasas/metabolismo , Xenopus/genéticaRESUMEN
Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/ß-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast, efficient, and penetrant genetic Xenopus tropicalis desmoid tumor models to identify and characterize drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 selection-mediated identification of dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double-strand break repair patterns to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1 as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent approach for in vivo mapping of tumor vulnerabilities and drug target identification.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/aislamiento & purificación , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Edición Génica/métodos , Neoplasias Abdominales/genética , Poliposis Adenomatosa del Colon/genética , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Fibromatosis Agresiva/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso , Oncogenes , Complejo Represivo Polycomb 2/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Xenopus , beta CateninaRESUMEN
The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.
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Anomalías Múltiples/etiología , Enfermedades del Desarrollo Óseo/etiología , Ectromelia/etiología , Cadera/anomalías , Homocigoto , Isquion/anomalías , Mutación con Pérdida de Función , Enfermedades Pulmonares/etiología , Pulmón/anomalías , Rótula/anomalías , Pelvis/anomalías , Proteínas de Dominio T Box/genética , Anomalías Múltiples/patología , Adolescente , Enfermedades del Desarrollo Óseo/patología , Niño , Ectromelia/patología , Femenino , Cadera/patología , Humanos , Isquion/patología , Pulmón/patología , Enfermedades Pulmonares/patología , Masculino , Rótula/patología , Linaje , Pelvis/patología , PronósticoRESUMEN
Caspases constitute a family of cysteine proteases centrally involved in programmed cell death, which is an integral part of normal embryonic and fetal development. However, it has become clear that specific caspases also have functions independent of cell death. In order to identify novel apoptotic and nonapoptotic developmental caspase functions, we designed and transgenically integrated novel fluorescent caspase reporter constructs in developing Xenopus embryos and tadpoles. This model organism has an external development, allowing direct and continuous monitoring. These studies uncovered a nonapoptotic role for the initiator caspase-9 in primitive blood formation. Functional experiments further corroborated that caspase-9, but possibly not the executioners caspase-3 and caspase-7, are required for primitive erythropoiesis in the early embryo. These data reveal a novel nonapoptotic function for the initiator caspase-9 and, for the first time, implicate nonapoptotic caspase activity in primitive blood formation.
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Caspasa 9/metabolismo , Xenopus laevis/sangre , Animales , Apoptosis/fisiología , Muerte Celular/fisiología , Diferenciación Celular/fisiología , Genes Reporteros , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Transducción de Señal , Transfección , Xenopus laevis/embriologíaRESUMEN
The evolutionarily conserved transmembrane anterior posterior transformation 1 protein, encoded by TAPT1, is involved in murine axial skeletal patterning, but its cellular function remains unknown. Our study demonstrates that TAPT1 mutations underlie a complex congenital syndrome, showing clinical overlap between lethal skeletal dysplasias and ciliopathies. This syndrome is characterized by fetal lethality, severe hypomineralization of the entire skeleton and intra-uterine fractures, and multiple congenital developmental anomalies affecting the brain, lungs, and kidneys. We establish that wild-type TAPT1 localizes to the centrosome and/or ciliary basal body, whereas defective TAPT1 mislocalizes to the cytoplasm and disrupts Golgi morphology and trafficking and normal primary cilium formation. Knockdown of tapt1b in zebrafish induces severe craniofacial cartilage malformations and delayed ossification, which is shown to be associated with aberrant differentiation of cranial neural crest cells.
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Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Anomalías Craneofaciales/genética , Proteínas de la Membrana/genética , Mutación/genética , Osificación Heterotópica/genética , Osteocondrodisplasias/genética , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Diferenciación Celular , Movimiento Celular , Cilios/metabolismo , Cilios/patología , Embrión no Mamífero/anomalías , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Masculino , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Cresta Neural/citología , Cresta Neural/metabolismo , Linaje , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Transducción de Señal , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
The speed by which clinical genomics is currently identifying novel potentially pathogenic variants is outperforming the speed by which these can be functionally (genotype-phenotype) annotated in animal disease models. However, over the past few years the emergence of CRISPR/Cas9 as a straight-forward genome editing technology has revolutionized disease modeling in vertebrate non-mammalian model organisms such as zebrafish, medaka and Xenopus. It is now finally possible, by CRISPR/Cas9, to rapidly establish clinically relevant disease models in these organisms. Interestingly, these can provide both cost-effective genotype-phenotype correlations for gene-(variants) and genomic rearrangements obtained from clinical practice, as well as be exploited to perform translational research to improve prospects of disease afflicted patients. In this review, we show an extensive overview of these new CRISPR/Cas9-mediated disease models and provide future prospects that will allow increasingly accurate modeling of human disease in zebrafish, medaka and Xenopus.
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Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Edición Génica , Animales , Marcación de Gen , Terapia Genética/métodos , Genómica , Mutación , Xenopus , Pez CebraRESUMEN
The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models. We believe that due to particular advantageous characteristics, outlined within this review, GEXM will become a valuable alternative animal model for modeling human cancer. Furthermore, we provide perspectives of how GEXM will be used as a platform for elucidation of novel therapeutic targets and for preclinical drug validation. Finally, we also discuss some future prospects on how the recent expansions and adaptations of the CRISPR/Cas9 toolbox might influence and push forward X. tropicalis cancer research.
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Sistemas CRISPR-Cas/genética , Ingeniería Genética , Neoplasias/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción/genética , Animales , Modelos Animales de Enfermedad , Marcación de Gen , Humanos , Neoplasias/patología , Xenopus/genéticaRESUMEN
Brain regionalisation, neuronal subtype diversification and circuit connectivity are crucial events in the establishment of higher cognitive functions. Here we report the requirement for the transcriptional repressor Fezf2 for proper differentiation of neural progenitor cells during the development of the Xenopus forebrain. Depletion of Fezf2 induces apoptosis in postmitotic neural progenitors, with concomitant reduction in forebrain size and neuronal differentiation. Mechanistically, we found that Fezf2 stimulates neuronal differentiation by promoting Wnt/ß-catenin signalling in the developing forebrain. In addition, we show that Fezf2 promotes activation of Wnt/ß-catenin signalling by repressing the expression of two negative regulators of Wnt signalling, namely lhx2 and lhx9. Our findings suggest that Fezf2 plays an essential role in controlling when and where neuronal differentiation occurs within the developing forebrain and that it does so by promoting local Wnt/ß-catenin signalling via a double-repressor model.
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Diferenciación Celular/fisiología , Neuronas/fisiología , Prosencéfalo/embriología , Factores de Transcripción/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Xenopus/metabolismo , Xenopus/embriología , beta Catenina/metabolismo , Análisis de Varianza , Animales , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ , Etiquetado Corte-Fin in Situ , Luciferasas , Microscopía Fluorescente , Factores de Transcripción/genética , Proteínas de Xenopus/genética , Dedos de ZincRESUMEN
BACKGROUND: Respiratory system development is regulated by a complex series of endoderm-mesoderm interactions that are not fully understood. Recently Xenopus has emerged as an alternative model to investigate early respiratory system development, but the extent to which the morphogenesis and molecular pathways involved are conserved between Xenopus and mammals has not been systematically documented. RESULTS: In this study, we provide a histological and molecular atlas of Xenopus respiratory system development, focusing on Nkx2.1+ respiratory cell fate specification in the developing foregut. We document the expression patterns of Wnt/ß-catenin, fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) signaling components in the foregut and show that the molecular mechanisms of respiratory lineage induction are remarkably conserved between Xenopus and mice. Finally, using several functional experiments we refine the epistatic relationships among FGF, Wnt, and BMP signaling in early Xenopus respiratory system development. CONCLUSIONS: We demonstrate that Xenopus trachea and lung development, before metamorphosis, is comparable at the cellular and molecular levels to embryonic stages of mouse respiratory system development between embryonic days 8.5 and 10.5. This molecular atlas provides a fundamental starting point for further studies using Xenopus as a model to define the conserved genetic programs controlling early respiratory system development.
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Embrión no Mamífero/embriología , Epistasis Genética/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Metamorfosis Biológica/fisiología , Sistema Respiratorio/embriología , Vía de Señalización Wnt/fisiología , Animales , Embrión no Mamífero/citología , Ratones , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Sistema Respiratorio/citología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Proteínas de Xenopus , Xenopus laevis , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/ß-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.
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Proliferación Celular , Proteínas Hedgehog/fisiología , Retina/embriología , Retina/crecimiento & desarrollo , Vía de Señalización Wnt/fisiología , Animales , Animales Modificados Genéticamente , Proliferación Celular/efectos de los fármacos , Antagonismo de Drogas , Embrión no Mamífero , Inhibidores Enzimáticos/farmacología , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Indoles/farmacología , Modelos Biológicos , Organogénesis/efectos de los fármacos , Organogénesis/genética , Organogénesis/fisiología , Oximas/farmacología , Receptor Cross-Talk/efectos de los fármacos , Receptor Cross-Talk/fisiología , Retina/efectos de los fármacos , Retina/metabolismo , Teratógenos/farmacología , Alcaloides de Veratrum/farmacología , Vía de Señalización Wnt/efectos de los fármacos , Xenopus laevis/embriologíaRESUMEN
Embryos and larvae of vertebrate species with external development are ideal subjects for investigating the dynamic spatiotemporal activity of developmental signaling pathways. The availability of efficient transgene technologies in Xenopus and zebrafish and the translucency and/or transparency of their embryos and larvae make these two species attractive for direct in vivo imaging of reporter gene expression. In this article we describe the design of efficient signaling reporters, using the Wnt/ß-catenin pathway as a representative example. We define methods for validating the reporter constructs and describe how they can be used to generate stable transgenic lines in Xenopus. We provide efficient methods used in our laboratory for raising the tadpoles and froglets rapidly to sexual maturity. We further discuss how the reporter lines can be used for delineating the dynamic activity of a signaling pathway and how modulators of the pathway can be scrutinized via chemical intervention and the micro-injection of synthetic RNAs or morpholinos. The strategic outline discussed in this paper provides a template for studying other developmental signaling pathways in Xenopus.
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Animales Modificados Genéticamente/metabolismo , Genes Reporteros , Ingeniería Genética/métodos , Vía de Señalización Wnt , Xenopus/genética , Animales , Desarrollo Embrionario/genética , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Organogénesis/genética , Xenopus/crecimiento & desarrollo , Xenopus/metabolismoRESUMEN
Cornified skin appendages, such as hair and nails, are major evolutionary innovations of terrestrial vertebrates. Human hair and nails consist largely of special intermediate filament proteins, known as hair keratins, which are expressed under the control of the transcription factor Hoxc13. Here, we show that the cornified claws of Xenopus frogs contain homologs of hair keratins and the genes encoding these keratins are flanked by promoters in which binding sites of Hoxc13 are conserved. Furthermore, these keratins and Hoxc13 are co-expressed in the claw-forming epithelium of frog toe tips. Upon deletion of hoxc13, the expression of hair keratin homologs is abolished and the development of cornified claws is abrogated in X. tropicalis. These results indicate that Hoxc13-dependent expression of hair keratin homologs evolved already in stem tetrapods, presumably as a mechanism for protecting toe tips, and that this ancestral genetic program was coopted to the growth of hair in mammals.
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Queratinas Específicas del Pelo , Factores de Transcripción , Animales , Humanos , Factores de Transcripción/metabolismo , Piel/metabolismo , Cabello/metabolismo , Queratinas/genética , Queratinas/metabolismo , Anfibios , Mamíferos/metabolismoRESUMEN
The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Células-Madre Neurales/citología , Retina/citología , Retina/embriología , Proteínas de Xenopus/biosíntesis , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ciclo Celular/fisiología , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Inmunohistoquímica , Masculino , Células-Madre Neurales/metabolismo , Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal , Vía de Señalización Wnt , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
The formation of primitive (embryonic) blood in vertebrates is mediated by spatio-temporally restricted signaling between different tissue layers. In Xenopus, in which primitive blood originates in the ventral blood island, this involves the secretion of bone morphogenetic protein (BMP) ligands by the ectoderm that signal to the underlying mesoderm during gastrulation. Using novel transgenic reporter lines, we report that the canonical Wnt/ß-catenin pathway is also activated in the blood islands in Xenopus. Furthermore, Wnt-reporter activity was also detected in the blood islands of the mouse yolk sac. By using morpholino-mediated depletion in Xenopus, we identified Wnt4 as the ligand that is expressed in the mesoderm of the ventral blood island and is essential for the expression of hematopoietic and erythroid marker genes. Injection of an inducible Wnt-interfering construct further showed that, during gastrulation, Wnt/ß-catenin signaling is required both in the mesoderm and in the overlying ectoderm for the formation of the ventral blood island. Using recombination assays with embryonic explants, we document that ectodermal BMP4 expression is dependent on Wnt4 signals from the mesoderm. Our results thus reveal a unique role for Wnt4-mediated canonical signaling in the formation and maintenance of the ventral blood island in Xenopus.
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Hematopoyesis , Transducción de Señal , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , beta Catenina/metabolismo , Animales , Proteína Morfogenética Ósea 4/genética , Proteína Morfogenética Ósea 4/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Wnt/genética , Proteína Wnt4 , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.
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Leucemia Mieloide Aguda , ARN Guía de Sistemas CRISPR-Cas , Animales , Humanos , Histona Metiltransferasas/genética , Xenopus/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , MutaciónRESUMEN
The Zic transcription factors play critical roles during embryonic development. Mutations in the ZIC2 gene are associated with human holoprosencephaly, but the etiology is still unclear. Here, we report a novel function for ZIC2 as a regulator of ß-catenin·TCF4-mediated transcription. We show that ZIC2 can bind directly to the DNA-binding high mobility group box of TCF4 via its zinc finger domain and inhibit the transcriptional activity of the ß-catenin·TCF4 complex. However, the binding of TCF4 to DNA was not affected by ZIC2. Zic2 RNA injection completely inhibited ß-catenin-induced axis duplication in Xenopus embryos and strongly blocked the ability of ß-catenin to induce expression of known Wnt targets in animal caps. Moreover, Zic2 knockdown in transgenic Xenopus Wnt reporter embryos led to ectopic Wnt signaling activity mainly at the midbrain-hindbrain boundary. Together, our results demonstrate a previously unknown role for ZIC2 as a transcriptional regulator of the ß-catenin·TCF4 complex.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/microbiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Embrión no Mamífero/metabolismo , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Factor de Transcripción 4 , Factores de Transcripción/genética , Proteínas Wnt/genética , Xenopus laevis , beta Catenina/genéticaRESUMEN
Lymph vessels control fluid homeostasis, immunity and metastasis. Unraveling the molecular basis of lymphangiogenesis has been hampered by the lack of a small animal model that can be genetically manipulated. Here, we show that Xenopus tadpoles develop lymph vessels from lymphangioblasts or, through transdifferentiation, from venous endothelial cells. Lymphangiography showed that these lymph vessels drain lymph, through the lymph heart, to the venous circulation. Morpholino-mediated knockdown of the lymphangiogenic factor Prox1 caused lymph vessel defects and lymphedema by impairing lymphatic commitment. Knockdown of vascular endothelial growth factor C (VEGF-C) also induced lymph vessel defects and lymphedema, but primarily by affecting migration of lymphatic endothelial cells. Knockdown of VEGF-C also resulted in aberrant blood vessel formation in tadpoles. This tadpole model offers opportunities for the discovery of new regulators of lymphangiogenesis.
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Linfangiogénesis/fisiología , Xenopus laevis/crecimiento & desarrollo , Xenopus laevis/genética , Animales , Proteínas de Homeodominio/fisiología , Larva/genética , Larva/crecimiento & desarrollo , Linfangiogénesis/genética , Sistema Linfático/anatomía & histología , Sistema Linfático/crecimiento & desarrollo , Proteínas Supresoras de TumorRESUMEN
The 22q11.2 deletion syndrome is a common dominant genetic disorder characterized by a heterozygous deletion of a cluster of genes on chromosome 22q11.2. TBX1, a transcription factor belonging to the T-box gene family, is a key player in the syndrome. However, heterozygosity of Tbx1 in mouse models does not fully recapitulate the phenotypes characteristic of the disease, which may point to the involvement of other genes in the deleted chromosomal region. Hence, we investigated the contribution of the catenin ARVCF, another gene that is deleted in 22q11.2DS. During Xenopus development, ARVCF mRNA is expressed in the pharyngeal arches and depleting either ARVCF or Tbx1 results in delayed migration of the cranial neural crest cells and in defects in the craniofacial skeleton and aortic arches. Moreover, double depletion of ARVCF and Tbx1 revealed that they act cooperatively, indicating that decreased ARVCF levels may also contribute to 22q11.2DS-associated phenotypes.