RESUMO
Dapper was isolated in a screen for proteins interacting with Dishevelled, a key factor in Wnt signaling. Dapper and Dishevelled colocalize intracellularly and form a complex with Axin, GSK-3, CKI, and beta-catenin. Overexpression of Dapper increases Axin and GSK-3 in this complex, resulting in decreased soluble beta-catenin and decreased activation of beta-catenin-responsive genes. Dapper also inhibits activation by Dishevelled of c-Jun N-terminal kinase (JNK), a component of beta-catenin-independent Frizzled signaling. Inhibition of Dapper activates both beta-catenin-responsive genes and an AP1-responsive promoter, demonstrating that Dapper is a general Dishevelled antagonist. Depletion of maternal Dapper RNA from Xenopus embryos results in loss of notochord and head structures, demonstrating that Dapper is required for normal vertebrate development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Notocorda/embriologia , Proteínas Nucleares , Fosfoproteínas/metabolismo , Proteínas Repressoras , Transativadores , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas de Peixe-Zebra , Animais , Proteína Axina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas de Transporte/química , Caseína Quinases , Sequência Conservada , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas Desgrenhadas , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase , Células HeLa , Humanos , Ligação de Hidrogênio , Técnicas In Vitro , MAP Quinase Quinase 4 , Dados de Sequência Molecular , Notocorda/metabolismo , Fenótipo , Ligação Proteica/fisiologia , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Homologia de Sequência de Aminoácidos , Vertebrados , Proteínas Wnt , Proteínas de Xenopus/química , Xenopus laevis , beta CateninaRESUMO
Transgene insertions in the mouse often cause mutations at chromosomal loci. Analysis of insertion mutations that cause male sterility may lead to the identification of novel molecular mechanisms implicated in male fertility. Here we show a line of transgenic mice with dominant inheritance of male sterility (DMS) that was found amid several lines that were normally fertile. Transgene-positive males from this line invariably were sterile, whereas transgenic females and transgene-negative male littermates were fertile. Histologic analysis and TUNEL staining for apoptotic cells in DMS testis showed spermatogenesis arrest at metaphase of meiosis I (M-I), accompanied by massive apoptosis of spermatocytes. Meiosis I arrest was incomplete, however, as small numbers of spermatids and spermatozoa were found. Both round spermatids and spermatozoa were evaluated for their permissiveness in the assisted reproductive technologies intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI). Surprisingly, ROSI but not ICSI gave live offspring, suggesting that mature sperm had deteriorated by the time of recovery from the epididymis. Mapping the transgene insertion by fluorescence in situ hybridization revealed a site on chromosome 14 D3-E1. Two candidate genes, GFR alpha 2 and GnRH, that were previously mapped to that region and the functions of which in spermatogenesis are well established were not altered in DMS. As a consequence, positional cloning of the DMS locus will be essential to identify new molecules potentially involved in arrest at M-I. Furthermore, mice carrying this genetic trait might be useful for studies of assisted reproductive technologies and male contraceptives.
