5849 Introduction Control of mRNA translation plays an important role in temporal and spatial control of gene expression during development in a variety of organisms (Wickens et al., 2000). Coupling of translational control to subcellular mRNA localization facilitates targeting and restriction of cytoplasmic proteins to specific cellular domains, and plays an essential role in the deployment of key patterning molecules in oocytes and embryos (Johnstone and Lasko, 2001). In the early Drosophila embryo, translation of nanos (nos) mRNA at the posterior pole produces a gradient of Nos protein that directs abdomen formation by repressing translation of maternal hunchback (hb) mRNA (Tautz, 1988; Gavis and Lehmann, 1992). Nos is also crucial at the posterior for germ cell function, by repressing translation of mRNAs like cyclinB (cycB) (Asaoka-Taguchi et al., 1999). Because Nos can also repress translation of the anteriorly localized bicoid (bcd) mRNA, however, it must be excluded from the anterior to allow head and thorax development (Wharton and Struhl, 1989; Gavis and Lehmann, 1992). Synthesis of Nos at the posterior of the embryo requires localization of maternal nos mRNA to the posteriorly localized germ plasm (Gavis and Lehmann, 1992; Wang et al., 1994). When localization of nos RNA is abolished by mutations in genes necessary for formation of the germ plasm, such as oskar (osk) and vasa (vas), nos translation is repressed and the resulting embryos lack abdominal segments (Gavis and Lehmann, 1994). Posterior localization of nos is inefficient, however, as the vast majority of nos RNA fails to become localized and is distributed throughout the embryo (Bergsten and Gavis, 1999). Translational repression of this unlocalized pool of nos mRNA is thus essential to restrict production of Nos protein to the posterior. A major theme in post-transcriptional regulation of developmentally relevant mRNAs is its reliance on cis-acting regulatory elements located within 3′ untranslated regions (3′UTRs) (Kuersten and Goodwin, 2003). The mechanisms by which many of these elements function are ill defined, however. Both posterior localization and translational repression of nos RNA require cis-acting sequences in the nos 3′UTR (Gavis and Lehmann, 1994). Translational repression of unlocalized nos is mediated by a 90 nucleotide translational control element (TCE), the function of which requires formation of two stem-loops (II and III) (Dahanukar and Wharton, 1996; Gavis et al., 1996; Smibert et al., 1996; Crucs et al., 2000). Stem-loop II contains a binding site for the Smaug (Smg) protein that has been designated as the Smaug Recognition Element (SRE) (Smibert et al., 1996; Crucs et al., 2000). Mutation of the SRE disrupts TCE function and loss of Smg results in ectopic nos activity, indicating that Smg is a repressor of nos translation (Dahanukar and Wharton, 1996; Smibert et al., 1996; Dahanukar et al., 1999). Although stem- loop III is also required for TCE function, existing evidence suggests that it acts independently of Smg. First, mutations that disrupt base pairing in stem-loop III disrupt TCE-mediated translational repression without affecting Smg binding. Second, the retention of TCE function when stem-loops II and III are separated by a large spacer suggests that the two regions of the TCE are recognized independently (Crucs et al., 2000). Translational control of gene expression plays a fundamental role in the early development of many organisms. In Drosophila, selective translation of nanos mRNA localized to the germ plasm at the posterior of the embryo, together with translational repression of nanos in the bulk cytoplasm, is essential for development of the anteroposterior body pattern. We show that both components to spatial control of nanos translation initiate during oogenesis and that translational repression is initially independent of Smaug, an embryonic repressor of nanos. Repression during oogenesis and embryogenesis are mediated by distinct stem loops within the nanos 3′ untranslated region; the Smaug-binding stem-loop acts strictly in the embryo, whereas a second stem-loop functions in the oocyte. Thus, independent regulatory modules with temporally distinct activities contribute to spatial regulation of nanos translation. We propose that nanos evolved to exploit two different stage-specific translational regulatory mechanisms. Key words: nanos, Translational control, Translational regulation, Translational repressor, Drosophila, Oogenesis, Embryogenesis, Maternal mRNA Summary Temporal complexity within a translational control element in the nanos mRNA Kevin M. Forrest, Ira E. Clark, Roshan A. Jain and Elizabeth R. Gavis* Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA *Author for correspondence (e-mail: lgavis@molbio.princeton.edu) Accepted 21 September 2004 Development 131, 5849-5857 Published by The Company of Biologists 2004 doi:10.1242/dev.01460 Research article