RESUMEN
Wnt signaling plays a pivotal role in embryogenesis and tissue homeostasis. Dishevelled (Dvl) is a central mediator for both Wnt/ß-catenin and Wnt/planar cell polarity pathways. NEDD4L, an E3 ubiquitin ligase, has been shown to regulate ion channel activity, cell signaling, and cell polarity. Here, we report a novel role of NEDD4L in the regulation of Wnt signaling. NEDD4L induces Dvl2 polyubiquitination and targets Dvl2 for proteasomal degradation. Interestingly, the NEDD4L-mediated ubiquitination of Dvl2 is Lys-6, Lys-27, and Lys-29 linked but not typical Lys-48-linked ubiquitination. Consistent with the role of Dvl in both Wnt/ß-catenin and Wnt/planar cell polarity signaling, NEDD4L regulates the cellular ß-catenin level and Rac1, RhoA, and JNK activities. We have further identified a hierarchical regulation that Wnt5a induces JNK-mediated phosphorylation of NEDD4L, which in turn promotes its ability to degrade Dvl2. Finally, we show that NEDD4L inhibits Dvl2-induced axis duplication in Xenopus embryos. Our work thus demonstrates that NEDD4L is a negative feedback regulator of Wnt signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/fisiología , Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Vía de Señalización Wnt , Secuencias de Aminoácidos , Animales , Secuencia Conservada , Proteínas Dishevelled , Retroalimentación Fisiológica , Células HEK293 , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ubiquitina-Proteína Ligasas Nedd4 , Unión Proteica , Estructura Terciaria de Proteína , Proteolisis , Ubiquitinación , Proteínas de Xenopus , Xenopus laevis/embriología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
To explore the effects of growth-related genes in both sexes and at different growth and development stages, male and female white Muscovy ducks at embryonic day E13, E17, E21, E25 and E29 were assessed in this study. RT-qPCR was used to determine the mRNA transcription levels of selected growth-related genes in the leg muscles of Muscovy ducks of both sexes and at different growth and developmental stages. MSTN, IGF2BP1 and FABP2 mRNAs were expressed in the leg muscles of male and female Muscovy ducks, but with different expression patterns. The MSTN and IGF2BP1 mRNA expression patterns were wavelike. MSTN mRNA expression was elevated at E13, increased at E17, decreased rapidly to the lowest level at E21, increased again at E25, and then decreased. IGF2BP1 mRNA expression was elevated at E13, increased at E17, decreased rapidly at E21, decreased rapidly to the lowest level at E25, and increased at E29. The expression trend of FABP2 mRNA was approximately "â¥" shape; the expression was the lowest at E13, increased slowly from E17 to E25, and increased extremely significantly at E29. In addition, the expression of MSTN in male Muscovy ducks was significantly higher than that in female ducks at E25 (P < 0.05). The expression of IGF2BP1 in male Muscovy ducks was extremely significantly higher than that in female ducks at E17 (P < 0.01). However, the expression of FABP2 in female Muscovy ducks was extremely significantly higher than that in male Muscovy ducks at E21 and E29 (P < 0.01). In conclusion, the mRNA expression of MSTN, IGF2BP1 and FABP2 in white Muscovy ducks is gestational age specific and sex specific. The differential gene expression patterns observed in this study provide a basis for understanding the physiological changes in white Muscovy ducks at different embryonic ages and in both sexes, supplementing the existing research on duck embryo muscle development. In addition, the findings provide a new framework for further discussion of poultry breeding.
Asunto(s)
Patos , Desarrollo de Músculos , Animales , Patos/genética , Patos/metabolismo , Desarrollo Embrionario , Femenino , Masculino , Músculo Esquelético/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Xmyf-5 gene is a member of the myogenic regulatory genes important for Xenopus muscle cell commitment and differentiation. To analyze the mechanism involved in regulating Xmyf-5 gene activity in the process of myogenesis, the 4.9 kb 5' upstream sequence of Xmyf-5 was identified. LacZ gene could be activated in the presomitic mesoderm in stage 14 embryos and myotomes in stage 25 embryos when it was driven by the 4.9 kb sequence and introduced into embryos at 2 cell stage. The Luciferase reporter gene activities driven by deletion constructs suggested that there are at least one enhancer between 4.9 kb and 3.5 kb of Xmyf-5 5' upstream DNA and one negatively regulative factor between 2.0 kb and 0.9 kb of the Xmyf-5 5' upstream DNA. In addition, sequence analysis indicated that a 100 bp fragment 5' upstream of Xmyf-5 contained conserved regulative elements such as OLS which were found in other vertebrates.