Although subcellular mRNA trafficking has been demonstrated as a mechanism to control protein distribution, it is generally believed that most protein localization occurs subsequent to translation. To address this point, we developed and employed a high-resolution fluorescent in situ hybridization procedure to comprehensively evaluate mRNA localization dynamics during early Drosophila embryogenesis. Surprisingly, of the 3370 genes analyzed, 71% of those expressed encode subcellularly localized mRNAs. Dozens of new and striking localization patterns were observed, implying an equivalent variety of localization mechanisms. Tight correlations between mRNA distribution and subsequent protein localization and function, indicate major roles for mRNA localization in nucleating localized cellular machineries. A searchable web resource documenting mRNA expression and localization dynamics has been established and will serve as an invaluable tool for dissecting localization mechanisms and for predicting gene functions and interactions.

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae , that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type–related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

Nuclear receptors are a family of transcription factors with structurally conserved ligand binding domains that regulate their activity. Despite intensive efforts to identify ligands, most nuclear receptors are still "orphans." Here, we demonstrate that the ligand binding pocket of the Drosophila nuclear receptor E75 contains a heme prosthetic group. E75 absorption spectra, resistance to denaturants, and effects of site-directed mutagenesis indicate a single, coordinately bound heme molecule. A correlation between the levels of E75 expression and the levels of available heme suggest a possible role as a heme sensor. The oxidation state of the heme iron also determines whether E75 can interact with its heterodimer partner DHR3, suggesting an additional role as a redox sensor. Further, the E75-DHR3 interaction is also regulated by the binding of NO or CO to the heme center, suggesting that E75 may also function as a diatomic gas sensor. Possible mechanisms and roles for these interactions are discussed.

Scalloped (Sd) and Vestigial (Vg) are each needed for Drosophila wing development. We show that Sd is required for Vg function and that altering their relative cellular levels inhibits wing formation. In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. Our observations indicate that Vg and Sd function coordinately to control the expression of genes required for wing development, which implies that Vg is a tissuespecific transcriptional intermediary factor of Sd. Received August 24, 1998; revised version accepted November 3, 1998. The Drosophila vestigial (vg) and scalloped (sd) genes are expressed in similar patterns during wing development, and mutations in either gene lead to loss of wing tissue (Campbell et al. 1991(Campbell et al. , 1992Williams et al. 1991Williams et al. , 1993. Vg is a developmentally regulated nuclear protein of previously unknown function and is required principally for the development of the wing and haltere (Williams et al. 1991). Sd is part of a highly conserved family of transcription factors, the TEA/ATTS domain proteins and is an essential protein with a wider developmental role (Campbell et al. 1991(Campbell et al. , 1992.Expression of vg in cells of the developing wing primordia is established by a number of conserved signaling pathways and is required for subsequent cell proliferation and patterning. Expression of wingless (wg), as well as interactions between dorsal and ventral cells that activate the Notch receptor, initially directs limited vg expression along the dorsal-ventral (D/V) wing boundary (Williams et al. 1994;Kim et al. 1995Kim et al. , 1996; for review, see Irvine and Vogt 1997). Subsequent vg expression in the wing primordia occurs in response to both the D/V Wg signal and Decapentaplegic (Dpp), a member of the Transforming Growth Factor-␤ (TGF-␤) protein family, secreted by cells along the anterior-posterior (A/P) border. (Blair 1994;Kim et al. 1996Kim et al. , 1997Zecca et al. 1996;Neumann and Cohen 1997). By the late third larval instar, maximal amounts of Vg are seen in cells at the D/V wing disc boundary, whereas cells located farther from this border produce progressively less Vg (Williams et al. 1991). vg is also required to maintain sd expression in the wing progenitor cells, and sd is similarly required for the maintenance of elevated vg expression (Williams et al. 1993).A cellular role for Sd can be inferred from studies of its human homolog Transcription Enhancer Factor-1 (TEF-1). TEF-1 binds to SV40 enhancer sequences via a TEA/ ATTS class DNA-binding domain, and has been shown to require transcriptional intermediary factors (TIFs) for proper function (Xiao et al. 1991;Ishiji et al. 1992;Hwang et al. 1993;Gupta et al. 1997). Interestingly, it has been reported that the Sd TEA/ATTS domain does not bind the same enhancer DNA sequences in vitro as TEF-1, although TEF-1...

Heme is a ligand for the human nuclear receptors (NR) REV-ERBα and REV-ERBβ, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 Å crystal structure of the REV-ERBβ LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBβ complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.