RESUMEN
Wilson's disease (WD) is a copper metabolic disorder caused by a defective ATP7B function. Conventional therapies cause severe side effects and significant variation in efficacy, according to cohort studies. Thus, exploring new therapeutic approaches to prevent progression to liver failure is urgent. To study the physiology and pathology of WD, immortalized cell lines and rodent WD models have been used conventionally; however, a large gap remains among different species as well as in genetic backgrounds among individuals. We generated induced pluripotent stem cells (iPSCs) from four WD patients carrying compound heterozygous mutations in the ATP7B gene. ATP7B loss- and gain-of-functions were further manifested with ATP7B-deficient iPSCs and heterozygously corrected R778L WD patient-derived iPSCs using CRISPR-Cas9-based gene editing. Although the expression of ATP7B protein varied among WD-specific hepatocytes differentiated from these iPSCs, the expression and secretion of ceruloplasmin (Cp), a downstream copper carrier in plasma, were consistently decreased in WD patient-derived and ATP7B-deficient hepatocytes. A transcriptome analysis detected abnormalities in the retinoid signaling pathway and lipid metabolism in WD-specific hepatocytes. Drug screening using WD patient-derived hepatocytes identified retinoids as promising candidates for rescuing Cp secretion. All-trans retinoic acid also alleviates reactive oxygen species production induced by lipid accumulation in WD-specific hepatocytes treated with oleic acid. These patient-derived iPSC-based hepatic models function as effective platforms for the development of potential therapeutics for hepatic steatosis in WD and other fatty liver diseases.
Asunto(s)
Degeneración Hepatolenticular , Humanos , Degeneración Hepatolenticular/tratamiento farmacológico , Degeneración Hepatolenticular/genética , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Cobre/metabolismo , Retinoides/metabolismo , Retinoides/uso terapéutico , ATPasas Transportadoras de Cobre/genética , Hepatocitos/metabolismo , Estrés Oxidativo , MutaciónRESUMEN
Dorsal-ventral pattern formation of the neural tube is regulated by temporal and spatial activities of extracellular signalling molecules. Sonic hedgehog (Shh) assigns ventral neural subtypes via activation of the Gli transcription factors. Shh activity in the neural progenitor cells changes dynamically during differentiation, but the mechanisms regulating this dynamicity are not fully understood. Here, we show that temporal change of intracellular cAMP levels confers the temporal Shh signal, and the purinergic G-protein-coupled receptor GPR17 plays an essential role in this regulation. GPR17 is highly expressed in the ventral progenitor regions of the neural tube and acts as a negative regulator of the Shh signal in chick embryos. Although the activation of the GPR17-related signal inhibits ventral identity, perturbation of Gpr17 expression leads to aberrant expansion of ventral neural domains. Notably, perturbation of Gpr17 expression partially inhibits the negative feedback of Gli activity. Moreover, GPR17 increases cAMP activity, suggesting that it exerts its function by inhibiting the processing of Gli3 protein. GPR17 also negatively regulates Shh signalling in neural cells differentiated from mouse embryonic stem cells, suggesting that GPR17 function is conserved among different organisms. Our results demonstrate that GPR17 is a novel negative regulator of Shh signalling in a wide range of cellular contexts.
Asunto(s)
Adaptación Fisiológica/fisiología , Proteínas Hedgehog/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Tubo Neural/embriología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Tipificación del Cuerpo/fisiología , Diferenciación Celular/genética , Embrión de Pollo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Desarrollo Embrionario/fisiología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Ratones , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal/genética , Transfección , Proteína Gli3 con Dedos de Zinc/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) are defined as multipotent cells that can give rise to various kinds of differentiated mesenchymal cells, and are thus considered to be useful for clinical therapy. However, the big hurdles of MSC therapy are the inability of MSCs to reach the appropriate tissues or sites with high efficiency and engraftment after transplantation. In this study, we investigated how adipose tissue-derived MSCs (AT-MSCs) improve their homing ability after intravenous injection. We previously found that human endothelial progenitor cells with low aldehyde dehydrogenase activity (Alde-Low EPCs) are suitable for the treatment of ischemic tissues. In addition, we demonstrated that microvesicles (MVs) derived from Alde-Low EPCs possessed the ability to improve the homing ability of non-functional Alde-High EPCs, resulting in wound healing. We initially transfected MVs derived from Alde-Low EPCs (EMVs) to human AT-MSCs, which were originally unable to cure ischemic tissues by intravenous transplantation. Remarkably, AT-MSC transfected EMVs dramatically repaired the ischemic skin flap compared with AT-MSC derived-MV (MMVs) transfected AT-MSCs or control AT-MSCs. We then found that the expression of CXCR4, an important chemokine receptor for cell migration, was highly elevated in EMV-transfected AT-MSCs. Moreover, AT-MSCs transfected with EMVs, but not control AT-MSCs, migrated to wound sites after intravenous injection. Consequently, CD45(+) inflammatory cells were successfully recruited at the wound sites after the injection of EMV-transfected AT-MSCs. These results demonstrate that EMVs are a useful source to improve the homing ability and wound healing ability of MSCs at the wound sites.
Asunto(s)
Células Progenitoras Endoteliales/citología , Células Madre Mesenquimatosas/citología , Cicatrización de Heridas , Animales , Ratones , Ratones Endogámicos C57BLRESUMEN
Juvenile nephronophthisis is an inherited renal ciliopathy with cystic kidney disease, renal fibrosis, and end-stage renal failure in children and young adults. Mutations in the NPHP1 gene encoding nephrocystin-1 protein have been identified as the most frequently responsible gene and cause the formation of cysts in the renal medulla. The molecular pathogenesis of juvenile nephronophthisis remains elusive, and no effective medicines to prevent end-stage renal failure exist even today. No human cellular models have been available yet. Here, we report a first disease model of juvenile nephronophthisis using patient-derived and gene-edited human induced pluripotent stem cells (hiPSCs) and kidney organoids derived from these hiPSCs. We established NPHP1-overexpressing hiPSCs from patient-derived hiPSCs and NPHP1-deficient hiPSCs from healthy donor hiPSCs. Comparing these series of hiPSCs, we found abnormalities in primary cilia associated with NPHP1 deficiency in hiPSCs. Kidney organoids generated from the hiPSCs lacking NPHP1 formed renal cysts frequently in suspension culture with constant rotation. This cyst formation in patient-derived kidney organoids was rescued by overexpression of NPHP1. Transcriptome analysis on these kidney organoids revealed that loss of NPHP1 caused lower expression of genes related to primary cilia in epithelial cells and higher expression of genes related to the cell cycle. These findings suggested the relationship between abnormality in primary cilia induced by NPHP1 loss and abnormal proliferative characteristics in the formation of renal cysts. These findings demonstrated that hiPSC-based systematic disease modeling of juvenile nephronophthisis contributed to elucidating the molecular pathogenesis and developing new therapies.
RESUMEN
Myelin basic protein (MBP) is a major component of the myelin sheaths of oligodendrocytes in the central nervous system and Schwann cells of the peripheral nervous system. Here we generated heterozygous fluorescent reporter of MBP gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock in fused tdTomato fluorescent protein and EF1 alpha promoter-driven Bleomycin (Zeocin) resistance gene to the translational MBP C-terminal region. The resulting line, MBP-TEZ, showed tdTomato fluorescence upon oligodendrocyte differentiation. This reporter hiPSC line provides a precedential opportunity for monitoring human myelin formation and degeneration and purifying MBP-expressing cell lineages.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Proteína Básica de Mielina/metabolismo , Proteína Básica de Mielina/genética , Vaina de Mielina/metabolismo , Línea Celular , Diferenciación Celular , Sistemas CRISPR-Cas , Genes Reporteros , Proteínas Luminiscentes/metabolismo , Proteínas Luminiscentes/genética , Edición Génica , Proteína Fluorescente RojaRESUMEN
Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome.
Asunto(s)
Células Madre Pluripotentes Inducidas , Proteína 2 de Unión a Metil-CpG , Síndrome de Rett , Síndrome de Rett/genética , Síndrome de Rett/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Femenino , Mutación , Línea Celular , Diferenciación CelularRESUMEN
During early embryogenesis, the neural plate is specified along the anterior-posterior (AP) axis by the action of graded patterning signals. In particular, the attenuation of canonical Wnt signals plays a central role in the determination of the anterior brain region. Here, we show that the extracellular matrix (ECM) protein Del1, expressed in the anterior neural plate, is essential for forebrain development in the Xenopus embryo. Overexpression of Del1 expands the forebrain domain and promotes the formation of head structures, such as the eye, in a Chordin-induced secondary axis. Conversely, the inhibition of Del1 function by a morpholino oligonucleotide (MO) represses forebrain development. Del1 also augments the expression of forebrain markers in neuralized animal cap cells, whereas Del1-MO suppresses them. We previously reported that Del1 interferes with BMP signaling in the dorsal-ventral patterning of the gastrula marginal zone. By contrast, we demonstrate here that Del1 function in AP neural patterning is mediated mainly by the inhibition of canonical Wnt signaling. Wnt-induced posteriorization of the neural plate is counteracted by Del1, and the Del1-MO phenotype (posteriorization) is reversed by Dkk1. Topflash reporter assays show that Del1 suppresses luciferase activities induced by Wnt1 and beta-catenin. This inhibitory effect of Del1 on canonical Wnt signaling, but not on BMP signaling, requires the Ror2 pathway, which is implicated in non-canonical Wnt signaling. These findings indicate that the ECM protein Del1 promotes forebrain development by creating a local environment that attenuates the cellular response to posteriorizing Wnt signals via a unique pathway.
Asunto(s)
Proteínas de la Membrana/metabolismo , Placa Neural/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Transducción de Señal , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Xenopus laevis/metabolismo , Transporte Activo de Núcleo Celular , Animales , Tipificación del Cuerpo , Cabeza/embriología , Proteínas de la Membrana/genética , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/genética , beta Catenina/metabolismoRESUMEN
DiGeorge syndrome (22q11.2 deletion syndrome, or CATCH22 syndrome), caused by hemizygous deletion of chromosome 22q11.2, results in the poor development of multiple organs. Here we have generated DiGeorge syndrome-specific human induced pluripotsnt stem cells (hiPSCs) derived from four patients. These established hiPSC lines showed self-renewal and pluripotency and carried a hemizygous deletion in 22q11.2. Since the molecular pathogenesis of DiGeorge syndrome caused by the 22q11.2 deletion is largely unknown, these cell resources will be useful for recapitulating disease phenotypes and for developing new therapies for DiGeorge syndrome.
Asunto(s)
Síndrome de DiGeorge , Células Madre Pluripotentes Inducidas , Cromosomas Humanos Par 2 , Síndrome de DiGeorge/genética , Humanos , FenotipoRESUMEN
Non-genetically modified somatic cells can only be inefficiently and stochastically reprogrammed to pluripotency by exogenous expression of reprogramming factors. Low competence of natural reprogramming factors may prevent the majority of cells to successfully and synchronously reprogram. Here we screened DNA-interacting amino acid residues in the zinc-finger domain of KLF4 for enhanced reprogramming efficiency using alanine-substitution scanning methods. Identified KLF4 L507A mutant accelerated and stabilized reprogramming to pluripotency in both mouse and human somatic cells. By testing all the variants of L507 position, variants with smaller amino acid residues in the KLF4 L507 position showed higher reprogramming efficiency. L507A bound more to promoters or enhancers of pluripotency genes, such as KLF5, and drove gene expression of these genes during reprogramming. Molecular dynamics simulations predicted that L507A formed additional interactions with DNA. Our study demonstrates how modifications in amino acid residues of DNA-binding domains enable next-generation reprogramming technology with engineered reprogramming factors.
RESUMEN
Adrenoleukodystrophy (ALD) is an X-linked genetic disorder, characterized by demyelination in the central nervous system and adrenal insufficiency. Human induced pluripotent stem cell (hiPSC) lines derived from two Japanese male patients with ALD were generated from skin fibroblasts using retroviral vectors. The generated hiPSC lines showed self-renewal and pluripotency, and carried either a missense or a nonsense mutation in ABCD1 gene. Since the molecular pathogenesis caused by ABCD1 dysfunction remains unclear, these cell resources provide useful tools to establish disease models and to develop new therapies for X-ALD.
Asunto(s)
Adrenoleucodistrofia , Enfermedades Genéticas Ligadas al Cromosoma X , Células Madre Pluripotentes Inducidas , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/genética , Adrenoleucodistrofia/genética , Fibroblastos , Humanos , Masculino , Mutación/genéticaRESUMEN
ISL1 encodes a member of the LIM/homeodomain family of transcription factors. This encoded protein plays central roles in the development of motor neuron, pancreas, and secondary heart field. Here we generated heterozygous fluorescent reporters of the ISL1 gene in human induced pluripotent stem cells (hiPSCs). CRISPR/Cas9 genome editing technology was employed to knock-in 2A-tdTomato and EF1 alpha promoter-driven Bleomycin resistance gene to the translational ISL1 C-terminal region. The resulting ISL1-TEZ lines showed tdTomato fluorescence upon motor neuron differentiation. These reporter iPSC lines provide opportunity for monitoring and purifying these related cell lineages.
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Edición Génica , Células Madre Pluripotentes Inducidas , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Proteínas Luminiscentes , Proteína Fluorescente RojaRESUMEN
Chromosomal abnormality causes congenital and acquired intractable diseases. In general, there are no fundamental treatments for these diseases. To establish platforms to develop therapeutics for these diseases, patient-derived induced pluripotent stem cells (iPSCs) are highly beneficial. To study abnormal chromosomal diseases, it is often hard to apply animal disease models because the chromosomal structures are variable among species. It is also difficult to apply simple genome editing technology in cells or individuals for abnormal chromosomes. Thus, these patient-derived iPSCs have advantages for developing disease models with multiple cell and tissue types, which are typically seen in the symptoms of abnormal chromosomal diseases. Here we review the studies of patient-derived iPSCs carrying abnormal chromosomes, focusing on pluripotent state and neural lineages. We also discuss the technological advances in chromosomal manipulations toward establishing experimental models and future therapeutics. Patient-derived iPSCs carrying chromosomal abnormality are valuable as cellular bioresources since they can indefinitely proliferate and provide various cell types. Also, these findings and technologies are important for future studies on elucidating pathogenesis, drug development, regenerative medicine, and gene therapy for abnormal chromosomal diseases.
RESUMEN
Juvenile nephronophthisis is an inherited renal ciliopathy, causing cystic kidney disease, renal fibrosis, and end-stage renal failure. Human induced pluripotent stem cell (hiPSC) lines, derived from two Juvenile nephronophthisis patients, were generated from peripheral blood mononuclear cells by episomal plasmid vectors. Generated hiPSC lines showed self-renewal and pluripotency and carried a large deletion in NPHP1 (Nephrocystin 1) gene. Since the molecular pathogenesis caused by NPHP1 dysfunction remains unclear, these cell resources provide useful tools to establish disease models and to develop new therapies for juvenile nephronophthisis.
Asunto(s)
Células Madre Pluripotentes Inducidas , Proteínas Adaptadoras Transductoras de Señales , Proteínas del Citoesqueleto , Fibrosis , Humanos , Enfermedades Renales Quísticas/congénito , Leucocitos Mononucleares , Proteínas de la Membrana/genéticaRESUMEN
We have isolated a novel secreted dorsalizing factor of the neural tube, Xenopus Tiarin, which belongs to the olfactomedin-related family. Tiarin expression starts at the late gastrula stage in the nonneural ectoderm adjacent to the anterior neural plate. Overexpression of Tiarin in the embryo causes expansion of dorsal neural markers and suppression of ventral markers. In the eye-forming field, Tiarin overexpression induces the retinal markers and represses optic stalk markers. Tiarin directly dorsalizes neural tissues in the absence of mesodermal tissues and antagonizes the ventralizing activity of Sonic hedghog (Shh). Unlike BMP4, another dorsalizing factor, Tiarin does not display antineuralizing activity on the ectoderm or mesoderm-ventralizing activity. These findings show that Tiarin is a novel patterning signal candidate acting in the specification of the dorsal neural tube.
Asunto(s)
Tipificación del Cuerpo/fisiología , Sistema Nervioso Central/embriología , Ectodermo/metabolismo , Inducción Embrionaria/fisiología , Sustancias de Crecimiento/metabolismo , Transducción de Señal/fisiología , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Diferenciación Celular/fisiología , Sistema Nervioso Central/metabolismo , Ectodermo/citología , Embrión no Mamífero , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Ojo/efectos de los fármacos , Ojo/embriología , Ojo/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Cabeza/embriología , Cabeza/fisiología , Mesodermo/citología , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Datos de Secuencia Molecular , Cresta Neural/efectos de los fármacos , Cresta Neural/embriología , Cresta Neural/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mensajero/farmacología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Xenopus laevis/metabolismoRESUMEN
Embryonic stem (ES) cells are useful for elucidating the molecular mechanisms of cell fate decision in the early development of mammals. It has been shown that aggregate culture of ES cells efficiently induces neuroectoderm differentiation. However, the molecular mechanism that leads to selective neural differentiation in aggregate culture is not fully understood. Here, we demonstrate that the oxygen-sensitive hypoxia-inducible transcription factor, Hif-1α, is an essential regulator for neural commitment of ES cells. We found that a hypoxic environment is spontaneously established in differentiating ES cell aggregates within 3 days, and that this time window coincides with Hif-1α activation. In ES cells in adherent culture under hypoxic conditions, Hif-1α activation was correlated with significantly greater expression of neural progenitor-specific gene Sox1 compared with ES cells in adherent culture under normoxic conditions. In contrast, Hif-1α-depleted ES cell aggregates showed severe reduction in Sox1 expression and maintained high expression of undifferentiated ES cell marker genes and epiblast marker gene Fgf5 on day 4. Notably, chromatin immune precipitation assay and luciferase assay showed that Hif-1α might directly activate Sox1 expression. Of additional importance is our finding that attenuation of Hif-1α resulted in an increase of BMP4, a potent inhibitor of neural differentiation, and led to a high level of phosphorylated Smad1. Thus, our results indicate that Hif-1α acts as a positive regulator of neural commitment by promoting the transition of ES cell differentiation from the epiblast into the neuroectoderm state via direct activation of Sox1 expression and suppressing endogenous BMP signaling.
Asunto(s)
Linaje de la Célula , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neuronas/citología , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Agregación Celular , Diferenciación Celular/genética , Hipoxia de la Célula/genética , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Modelos Biológicos , Placa Neural/citología , Neuronas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de Señal/genéticaRESUMEN
The generation of induced pluripotent stem cells (iPSCs) by introducing reprogramming factors into somatic cells is a promising method for stem cell therapy in regenerative medicine. Therefore, it is desirable to develop a minimally invasive simple method to create iPSCs. In this study, we generated human nasal epithelial cells (HNECs)-derived iPSCs by gene transduction with Sendai virus (SeV) vectors. HNECs can be obtained from subjects in a noninvasive manner, without anesthesia or biopsy. In addition, SeV carries no risk of altering the host genome, which provides an additional level of safety during generation of human iPSCs. The multiplicity of SeV infection ranged from 3 to 4, and the reprogramming efficiency of HNECs was 0.08-0.10%. iPSCs derived from HNECs had global gene expression profiles and epigenetic states consistent with those of human embryonic stem cells. The ease with which HNECs can be obtained, together with their robust reprogramming characteristics, will provide opportunities to investigate disease pathogenesis and molecular mechanisms in vitro, using cells with particular genotypes.
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Reprogramación Celular , Vectores Genéticos/genética , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mucosa Nasal/citología , Virus Sendai/genética , Transducción Genética/métodos , Femenino , HumanosRESUMEN
Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.
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Antígenos de Diferenciación/metabolismo , Tipificación del Cuerpo/fisiología , Corteza Cerebral/citología , Células Madre Embrionarias/metabolismo , Neuronas/metabolismo , Animales , Proteína Morfogenética Ósea 4/metabolismo , Ciclo Celular , Diferenciación Celular , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/trasplante , Factor 8 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Homeostasis , Humanos , Inmunohistoquímica , Ratones , Neuronas/citología , Neuronas/trasplante , Transducción de Señal , Técnicas de Cultivo de Tejidos , Proteínas Wnt/metabolismo , Proteína Wnt3RESUMEN
In Xenopus, an asymmetric distribution of Wnt activity that follows cortical rotation in the fertilized egg leads to the dorsal-ventral (DV) axis establishment. However, how a clear DV polarity develops from the initial difference in Wnt activity still remains elusive. We report here that the Teashirt-class Zn-finger factor XTsh3 plays an essential role in dorsal determination by enhancing canonical Wnt signaling. Knockdown of the XTsh3 function causes ventralization in the Xenopus embryo. Both in vivo and in vitro studies show that XTsh3 substantially enhances Wnt signaling activity in a beta-catenin-dependent manner. XTsh3 cooperatively promotes the formation of a secondary axis on the ventral side when combined with weak Wnt activity, whereas XTsh3 alone has little axis-inducing ability. Furthermore, Wnt1 requires XTsh3 for its dorsalizing activity in vivo. Immunostaining and protein analyses indicate that XTsh3 is a nuclear protein that physically associates with beta-catenin and efficiently increases the level of beta-catenin in the nucleus. We discuss the role of XTsh3 as an essential amplifying factor of canonical Wnt signaling in embryonic dorsal determination.
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Proteínas Nucleares/fisiología , Proteínas Wnt/metabolismo , Proteínas de Xenopus/fisiología , Xenopus/fisiología , beta Catenina/metabolismo , Animales , Tipificación del Cuerpo , Embrión no Mamífero/fisiología , Unión Proteica , Transducción de Señal , Xenopus/embriología , Xenopus/metabolismo , Dedos de ZincRESUMEN
We show here that a secreted EGF-Discoidin-domain protein, Xenopus Del1 (xDel1), is an essential factor for dorsal development in the early Xenopus embryo. Knockdown of the xDel1 function causes obvious ventralization of the embryo. Conversely, overexpression of xDel1 expands dorsal-marker expression and suppresses ventral-marker expression in the gastrula embryo. Forced expression of xDel1 dorsalizes ventral marginal zone explants, whereas it weakly induces neural differentiation but not mesodermal differentiation in animal caps. The dorsalizing activity of xDel1 is dependent on the Discoidin domains and not on the RGD motif (which is implicated in its angiogenic activity) or EGF repeats. Luciferase assays show that xDel1 attenuates BMP-signaling reporter activity by interfering with the pathway downstream of the BMP receptor. Thus, xDel1 functions as a unique extracellular regulatory factor of DV patterning in early vertebrate embryogenesis.
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Desarrollo Embrionario/genética , Proteínas de la Membrana/fisiología , Proteínas de Xenopus/fisiología , Xenopus/embriología , Secuencias de Aminoácidos , Animales , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/metabolismo , Embrión no Mamífero/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Oligopéptidos/química , Estructura Terciaria de Proteína , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genéticaRESUMEN
Here, we report in vitro generation of Math1+ cerebellar granule cell precursors and Purkinje cells from ES cells by using soluble patterning signals. When neural progenitors induced from ES cells in a serum-free suspension culture are subsequently treated with BMP4 and Wnt3a, a significant proportion of these neural cells become Math1+. The induced Math1+ cells are mitotically active and express markers characteristic of granule cell precursors (Pax6, Zic1, and Zipro1). After purification by FACS and coculture with postnatal cerebellar neurons, ES cell-derived Math1+ cells exhibit typical features of neurons of the external granule cell layer, including extensive motility and a T-shaped morphology. Interestingly, differentiation of L7+/Calbindin-D28K+ neurons (characteristic of Purkinje cells) is induced under similar culture conditions but exhibits a higher degree of enhancement by Fgf8 rather than by Wnt3a. This is the first report of in vitro recapitulation of early differentiation of cerebellar neurons by using the ES cell system.