Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Here, using genetic and chemical manipulations, we show that nitric oxide is produced in the Drosophila prothoracic gland, where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates.[Keywords: E75; DHR3; nitric oxide; Drosophila; metamorphosis; ecdysone; metabolism] Supplemental material is available for this article. In previous work, we showed that ecdysone-induced protein 75 (E75, also known as Eip75B; NR1D3) (Tweedie et al. 2009) contains heme constitutively bound to its ligand-binding domain (LBD), and that amino acids coordinately bound to the heme iron can be displaced in vitro by changes in redox state or the presence of nitric oxide (NO) gas (Pardee et al. 2004;Reinking et al. 2005;Marvin et al. 2009). In turn, these structural changes negate the ability of E75 to repress transcription and to reverse the positive transcriptional activity of its heterodimer partner, Drosophila hormone receptor 3 (DHR3; also known as DHR46, NR1F4) (Tweedie et al. 2009). Here, we look to see whether these interactions are relevant in vivo and, if so, what their roles are.One of the best-characterized roles of E75 and DHR3 in vivo is within the nuclear receptor (NR) transcriptional hierarchy that controls, and responds to, the production of the metamorphosis-inducing hormone ecdysone (diagrammed in Supplemental Fig. 1A). Upon binding ecdysone, the ecdysone receptor (EcR) acts as a heterodimer with a second NR, ultraspiracle (USP), to activate transcription of DHR3 and the E75 isoform E75A (Koelle et al. 1991;Lam et al. 1997Lam et al. , 1999White et al. 1997;Bialecki et al. 2002). DHR3 then promotes its own continued expression as well as that of the E75 splice variant E75B and the downstream NR gene bFtz-F1. bFTZ-F1, in turn, activates the expression of ecdysone synthetic enzyme genes (Lavorgna et al. 1993;Woodard et al. 1994;Broadus et al. 1999), resulting in another round of ecdysone production. The major site of larval ecdysone production is the prothoracic gland (PG) (diagrammed in Supplemental Fig. 1B). The PG is also a major site of NO synthase (Nos) expression (Wildemann and Bicker 1999). Hence, we looked to see whether E75 and NO are present and interactive in this tissue, with the hypothesis that activation of bFtz-F1 transcription by DHR3 requires inactivation of E75 isoforms by NO. As predicted, all of the above-listed genes are expressed in the PG toward the end of third instar development, and disruption of their expression or activity leads to molec...

The asymmetric localization of gurken mRNA and protein in the developing Drosophila oocyte defines both the anteroposterior and dorsoventral axes of the future embryo. Understanding the origin of these asymmetries requires knowledge of the source of gurken transcripts. During oogenesis most transcripts in the oocyte are produced by the associated nurse cells, but it has been proposed that gurken is an exceptional oocyte-derived transcript. Using a novel application of a standard mitotic recombination technique, we generated mosaic egg chambers in which the nurse cells, but not the oocyte, could produce gurken. Gurken was properly localized in these mosaics and oocyte axial polarity was established normally,indicating that the nurse cells synthesize gurken and that their contribution is sufficient for Gurken function. Our data demonstrate the existence of a mechanism for transport of gurken from the nurse cells and its subsequent localization within the oocyte.

INTRODUCTIONFluorescent in situ hybridization (FISH) is commonly used to analyze the three-dimensional distribution of RNAs in intact embryos and tissues. Tyramide signal amplification (TSA) significantly increases the sensitivity and resolution of FISH probe signals. This protocol includes optimized TSA-FISH procedures for Drosophila embryos, ovaries, and larval tissues. Instructions are given for the preparation of RNA probes, the collection and fixation of tissues, and the hybridization and TSA-mediated detection of probes, including options for high-throughput processing in 96-well plates. Variations of the procedure for RNA-RNA and RNA-protein costaining are also described.

Establishment of the Drosophila dorsal-ventral axis depends upon the correct localization of gurken mRNA and protein within the oocyte. gurken mRNA becomes localized to the presumptive dorsal anterior region of the oocyte, but is synthesized in the adjoining nurse cells. Normal gurken localization requires the heterogeneous nuclear ribonucleoprotein Squid, which binds to the gurken 3' untranslated region. However, whether Squid functions in the nurse cells or the oocyte is unknown. To address this question, we generated genetic mosaics in which half of the nurse cells attached to a given oocyte are unable to produce Squid. In these mosaics, gurken mRNA is localized normally but ectopically translated during the dorsal anterior localization process, even though the oocyte contains abundant Squid produced by the wild type nurse cells. These data indicate that translational repression of gurken mRNA requires Squid function in the nurse cells. We propose that Squid interacts with gurken mRNA in the nurse cell nuclei and, together with other factors, maintains gurken in a translationally silent state during its transport to the dorsal anterior region of the oocyte. This translational repression is not required for gurken mRNA localization, indicating that the information repressing translation is separable from that regulating localization.

We review recent trends to improve the precision and efficiency of oligo-based point mutation knock-ins and discuss how these improvements can apply to work in zebrafish. Similarly to how chemical mutagenesis enabled the first genetic screens in zebrafish, multiplexed sgRNA libraries and Cas9 can enable the next revolutionary transition in how genetic screens are performed in this species. We discuss the first examples and prospects of approaches using sgRNAs as specific and effective mutagens. Moreover, we have reviewed methods aimed at measuring the phenotypes of single cells after their mutagenic perturbation with vectors encoding individual sgRNAs. These methods can range from different cell-based reporters to single-cell RNA sequencing and can serve as great tools for high-throughput genetic screens.