RESUMEN
Although the zinc finger transcription factor Wt1 has been linked to female fertility, its precise role in this process has not yet been understood. We have sequenced the WT1 exons in a panel of patients with idiopathic infertility and have identified a missense mutation in WT1 in one patient out of eight. This mutation leads to an amino acid change within the zinc finger domain and results in reduced DNA binding. We utilized Wt1+/- mice as a model to mechanistically pinpoint the consequences of reduced Wt1 levels for female fertility. Our results indicate that subfertility in Wt1+/- female mice is a maternal effect caused by the Wt1-dependent de-regulation of Prss29, encoding a serine protease. Notably, blocking Prss29 activity was sufficient to rescue subfertility in Wt1+/- mice indicating Prss29 as a critical factor in female fertility. Molecularly, Wt1 represses expression of Prss29. De-repression and precocious expression of Prss29 in the oviduct of Wt1+/- mice interferes with pre-implantation development. Our study reveals a novel role for Wt1 in early mammalian development and identifies proteases as critical mediators of the maternal-embryonic interaction. Our data also suggest that the role of Wt1 in regulating fertility is conserved in mammals.
Asunto(s)
Infertilidad Femenina/genética , Proteínas WT1/genética , Proteínas WT1/metabolismo , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Exones , Femenino , Fertilidad/fisiología , Humanos , Infertilidad Femenina/sangre , Infertilidad Femenina/metabolismo , Ratones , Ratones Noqueados , Mutación Missense , Oviductos/metabolismo , Oviductos/patología , Factores de Transcripción/genética , Dedos de ZincRESUMEN
The Wilms' tumor suppressor gene 1 (WT1) encodes a zinc finger transcription factor. Mutation of WT1 in humans leads to Wilms' tumor, a pediatric kidney tumor, or other kidney diseases, such as Denys-Drash and Frasier syndromes. We showed previously that inactivation of WT1 in podocytes of adult mice results in proteinuria, foot process effacement, and glomerulosclerosis. However, the WT1-dependent transcriptional network regulating podocyte development and maintenance in vivo remains unknown. Here, we performed chromatin immunoprecipitation followed by high-throughput sequencing with glomeruli from wild-type mice. Additionally, we performed a cDNA microarray screen on an inducible podocyte-specific WT1 knockout mouse model. By integration of cistromic and transcriptomic analyses, we identified the WT1 targetome in mature podocytes. To further analyze the function and targets of WT1 in podocyte maturation, we used an Nphs2-Cre model, in which WT1 is deleted during podocyte differentiation. These mice display anuria and kidney hemorrhage and die within 24 hours after birth. To address the evolutionary conservation of WT1 targets, we performed functional assays using zebrafish as a model and identified Nphs2, Mafb, and Magi2 as novel WT1 target genes required for podocyte development. Our data also show that both Mafb and Magi2 are required for normal development of the embryonic zebrafish kidney. Collectively, our work provides insights into the transcriptional networks controlled by WT1 and identifies novel WT1 target genes that mediate the function of WT1 in podocyte differentiation and maintenance.
Asunto(s)
Diferenciación Celular/genética , Regulación de la Expresión Génica , Genes del Tumor de Wilms/fisiología , Podocitos/fisiología , Proteínas Represoras/genética , Proteínas WT1/genética , Proteínas de Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Perfilación de la Expresión Génica , Guanilato-Quinasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Factor de Transcripción MafB/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Pez CebraRESUMEN
The Wilms' tumor protein Wt1 plays an essential role in mammalian urogenital development. WT1 mutations in humans lead to a variety of disorders, including Wilms' tumor, a pediatric kidney cancer, as well as Frasier and Denys-Drash syndromes. Phenotypic anomalies in Denys-Drash syndrome include pseudohermaphroditism and sex reversal in extreme cases. We have used cDNA microarray analyses on Wt1 knockout mice to identify Wt1-dependent genes involved in sexual development. The gene most dramatically affected by Wt1 inactivation was Amhr2, encoding the anti-Müllerian hormone (Amh) receptor 2. Amhr2 is an essential factor for the regression of the Müllerian duct in males, and mutations in AMHR2 lead to the persistent Müllerian duct syndrome, a rare form of male pseudohermaphroditism. Here we show that Wt1 and Amhr2 are coexpressed during urogenital development and that the Wt1 protein binds to the promoter region of the Amhr2 gene. Inactivation and overexpression of Wt1 in cell lines was followed by immediate changes of Amhr2 expression. The identification of Amhr2 as a Wt1 target provides new insights into the role of Wt1 in sexual differentiation and indicates, in addition to its function in early gonad development and sex determination, a novel function for Wt1, namely, in Müllerian duct regression.
Asunto(s)
Genes del Tumor de Wilms , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Proteínas WT1/metabolismo , Tumor de Wilms/genética , Animales , Sitios de Unión , Línea Celular , Inmunoprecipitación de Cromatina , ADN Complementario , Genes Reporteros , Luciferasas/metabolismo , Masculino , Enfermedades Urogenitales Masculinas/genética , Enfermedades Urogenitales Masculinas/patología , Mesonefro/citología , Mesonefro/metabolismo , Ratones , Ratones Noqueados , Modelos Genéticos , Conductos Paramesonéfricos/embriología , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Unión Proteica , Receptores de Factores de Crecimiento Transformadores beta , Células de Sertoli/metabolismo , Proteínas WT1/genéticaRESUMEN
Mutations in the human EYA1 gene have been associated with several human diseases including branchio-oto (BO) and branchio-oto-renal (BOR) syndrome, as well as congenital cataracts and ocular anterior segment anomalies. BOR patients suffer from severe malformations of the ears, branchial arches and kidneys. The phenotype of Eya1-heterozygous mice resembles the symptoms of human patients suffering from BOR syndrome. The Eya1 gene encodes a multifunctional protein that acts as a protein tyrosine phosphatase and a transcriptional coactivator. It has been shown that Eya1 interacts with Six transcription factors, which are also required for nuclear translocation of the Eya1 protein. We investigated the effects of seven disease-causing Eya1 missense mutations on Eya1 protein function, in particular cellular localization, ability to interact with Six proteins, and protein stability. We show here that the BOR-associated Eya1 missense mutations S454P, L472R, and L550P lead to enhanced proteasomal degradation of the Eya1 protein in mammalian cells. Moreover, Six proteins lead to a significant stabilization of Eya1, which is caused by Six-mediated protection from proteasomal degradation. In case of the mutant L550P, loss of interaction with Six proteins leads to rapid protein degradation. Our observations suggest that protein destabilization constitutes a novel disease causing mechanism for Eya1.
Asunto(s)
Síndrome Branquio Oto Renal/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Tirosina Fosfatasas/genética , Animales , Células COS , Línea Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Mutación Missense , Proteínas Nucleares/metabolismo , Unión Proteica , Estabilidad Proteica , Transporte de Proteínas , Proteínas Tirosina Fosfatasas/metabolismo , Proteolisis , UbiquitinaciónRESUMEN
The eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome, characterized by kidney defects, hearing loss, and branchial arch anomalies. For a better understanding of Eya1's function, we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as novel interaction partners of Eya1. We confirmed the interactions by glutathione S-transferase (GST) pulldown analysis and coimmunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Using reverse transcriptase PCR (RT-PCR) and in situ hybridization, we show that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. By morpholino-mediated knockdown, we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 ortholog as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype, with characteristic defects in ear and branchial arch formation.
Asunto(s)
Proteínas Portadoras/metabolismo , Cabeza , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Síndrome Branquio Oto Renal/genética , Proteínas Portadoras/genética , Línea Celular , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Embrión no Mamífero/anatomía & histología , Embrión no Mamífero/fisiología , Cabeza/anatomía & histología , Cabeza/embriología , Cabeza/crecimiento & desarrollo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Fenotipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Distribución Tisular , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
microRNAs (miRNAs) are small non-coding RNAs with fundamental roles in the regulation of gene expression. miRNAs assemble with Argonaute (Ago) proteins to miRNA-protein complexes (miRNPs), which interact with distinct binding sites on mRNAs and regulate gene expression. Specific miRNAs are key regulators of tissue and organ development and it has been shown in mammals that miRNAs are also involved in the pathogenesis of many diseases including cancer. Here, we have characterized the miRNA expression profile of the developing murine genitourinary system. Using a computational approach, we have identified several miRNAs that are specific for the analyzed tissues or the developmental stage. Our comprehensive miRNA expression atlas of the developing genitourinary system forms an invaluable basis for further functional in vivo studies.
Asunto(s)
Perfilación de la Expresión Génica , MicroARNs/genética , Sistema Urogenital/crecimiento & desarrollo , Sistema Urogenital/metabolismo , Animales , Secuencia de Bases , Análisis por Conglomerados , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE(-/-) mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin beta receptor (LTbetaR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE(-/-) mice with LTbetaR-Ig to interrupt LTbetaR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTbetaR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.