RESUMEN
The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.
Asunto(s)
Proteínas de Unión al Calcio/genética , Embrión no Mamífero/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas Hedgehog/genética , Transducción de Señal/genética , Proteínas de Pez Cebra/genética , Animales , Western Blotting , Células COS , Proteínas de Unión al Calcio/metabolismo , Chlorocebus aethiops , ADN Complementario/química , ADN Complementario/genética , Embrión no Mamífero/embriología , Proteínas de la Matriz Extracelular/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Hedgehog/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Fenotipo , Análisis de Secuencia de ADN , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.