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The foraging (for) gene of Drosophila melanogaster encodes a cGMP-dependent protein kinase (PKG), which is a major effector of the cGMP signaling pathway involved in the regulation of behaviour and metabolic traits. Despite being well studied at the transcript level, little is known about the for gene at the protein level. Here, we provide a detailed characterization of the for gene protein (FOR) products and present new tools for their study, including five isoform-specific antibodies and a transgenic strain that carries an HA-labelled for allele (forBAC::HA). Our results showed that multiple FOR isoforms were expressed in the larval and adult stages of D. melanogaster and that the majority of whole-body FOR expression arises from three (P1, P1α, and P3) of eight putative protein isoforms. We found that FOR expression differed between the larval and adult stages and between the dissected larval organs we analyzed, which included the central nervous system (CNS), fat body, carcass, and intestine. Moreover, we showed that the FOR expression differed between two allelic variants of the for gene, namely, fors (sitter) and forR (rover), that are known to differ in many food-related traits. Together, our in vivo identification of FOR isoforms and the existence of temporal, spatial, and genetic differences in their expression lay the groundwork for determining their functional significance.

Proper differentiation of sperm from germline stem cells, essential for production of the next generation, requires dramatic changes in gene expression that drive remodeling of almost all cellular components, from chromatin to organelles to cell shape itself. Here we provide a single nucleus and single cell RNA-seq resource covering all of spermatogenesis inDrosophilastarting from in-depth analysis of adult testis single nucleus RNA-seq (snRNA-seq) data from the Fly Cell Atlas (FCA) study (Liet al., 2022). With over 44,000 nuclei and 6,000 cells analyzed, the data provide identification of rare cell types, mapping of intermediate steps in differentiation, and the potential to identify new factors impacting fertility or controlling differentiation of germline and supporting somatic cells. We justify assignment of key germline and somatic cell types using combinations of known markers,in situhybridization, and analysis of extant protein traps. Comparison of single cell and single nucleus datasets proved particularly revealing of dynamic developmental transitions in germline differentiation. To complement the web-based portals for data analysis hosted by the FCA, we provide datasets compatible with commonly used software such as Seurat and Monocle. The foundation provided here will enable communities studying spermatogenesis to interrogate the datasets to identify candidate genes to test for functionin vivo.

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is emerging as an important target in the brain for the treatment or prevention of cognitive disorders. The identification of high-affinity ligands for brain PPARα may reveal the mechanisms underlying the synaptic effects of this receptor and facilitate drug development. Here, using an affinity purification-untargeted mass spectrometry (AP-UMS) approach, we identified an endogenous, selective PPARα ligand, 7(

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amidst limited arable land. Most traditional nematicides have been banned due to poor nematode-selectivity, leaving farmers with inadequate controls. Here, we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome p450-dependent bioactivation exclusively in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita – the world’s most destructive plant-parasitic nematode. Tests against a wide range of phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than nearly all marketed nematicides. Thus, selectivins are first-in-class bioactivated nematode controls that provide efficacy as well as much-needed nematode selectivity.

Brominated azo dyes (BADs) have been identified as predominant indoor brominated pollutants in daycare dust; thus, their potential health risk to children is of concern. However, the toxicities of BADs remain elusive. In this study, the toxicokinetics of two predominant BADs, Disperse Blue 373 (DB373) and Disperse Violet 93 (DV93), and their suspect metabolite 2-bromo-4,6-dinitroaniline (BDNA) was investigated in embryos of zebrafish (Danio rerio). The bioconcentration factor of DV93 at 120 hpf is 6.2-fold lower than that of DB373. The nontarget analysis revealed distinct metabolism routes between DB373 and DV93 by reducing nitro groups to nitroso (DB373) or amine (DV93), despite their similar structures. NAD(P)H quinone oxidoreductase 1 (NQO1) and pyruvate dehydrogenase were predicted as the enzymes responsible for the reduction of DB373 and DV93 by correlating time courses of the metabolites and enzyme development. Further in vitro recombinant enzyme and in vivo inhibition results validated NQO1 as the enzyme specifically reducing DB373, but not DV93. Global proteome profiling revealed that the expression levels of proteins from the "apoptosis-induced DNA fragmentation" pathway were significantly upregulated by all three BADs, supporting the bioactivation of BADs to mutagenic aromatic amines. This study discovered the bioactivation of BADs via distinct eukaryotic enzymes, implying their potential health risks.

We have investigated the relationship between the function of the genehindsight(hnt), which is the Drosophila homolog ofRas Responsive Element Binding protein-1(RREB-1), and the EGFR signaling pathway. We report thathntmutant embryos are defective in EGFR signaling dependent processes, namely chordotonal organ recruitment and oenocyte specification. We also show the temperature sensitive hypomorphic allelehntpebbledis enhanced by the hypomorphic MAPK allelerolled(rl1). We find thathntoverexpression results in ectopicDPax2expression within the embryonic peripheral nervous system, and we show that this effect is EGFR-dependent. Finally, we show that the canonical U-shaped embryonic lethal phenotype ofhnt,which is associated with premature degeneration of the extraembyonic amnioserosa and a failure in germ band retraction, is rescued by expression of several components of the EGFR signaling pathway (sSpi,Ras85DV12,pntP1) as well as the caspase inhibitorp35. Based on this collection of corroborating evidence, we suggest that an overarching function ofhntinvolves the positive regulation of EGFR signaling.

Our recent ability to sequence entire genomes, along with all of their transcribed RNAs, has led to the surprising finding that only ∼1% of the human genome is used to encode proteins. This finding has led to vigorous debate over the functional importance of the transcribed but untranslated portions of the genome. Currently, scientists tend to assume coding genes are functional until proven not to be, while the opposite is true for noncoding genes. This review takes a new look at the evidence for and against widespread noncoding gene functionality. We focus in particular on long noncoding RNA (noncoding RNAs longer than 200 nucleotides) genes and their 'junk' associates, transposable elements, and satellite repeats. Taken together, the suggestion put forward is that more of this junk DNA may be functional than nonfunctional and that noncoding RNAs and transposable elements act symbiotically to drive evolution.

Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 via scRNA-seq and RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/., Author summary Male flies need to boost gene expression from their single X chromosome to equal that of females, which have two X chromosomes. In this process, called dosage compensation, the dosage compensation complex binds to genomic chromatin entry sites and upregulates gene expression nearby. This process was thought to be restricted to somatic cells. Using single-cell RNA-seq data, we found that certain germ cell types in the Drosophila testis show X chromosome expression similar to that of the autosomes, implying dosage compensation activity. In these cell types, we found evidence that genes near a chromatin entry site are more highly expressed than genes farther away, which is additional evidence of dosage compensation. In cell types without evidence of dosage compensation, we saw no evidence of chromatin entry site activity. Interestingly, we found little evidence of expression of most genes from the dosage compensation complex using both RNA-FISH and scRNA-seq. This suggests that our observed pre-meiotic dosage compensation is likely to be mediated by a noncanonical mechanism. These findings add new insight into our understanding of sex chromosomes.

Background: Thousands of per- and polyfluoroalkyl substances (PFAS) with diverse structures have been detected in the ambient environment. Apart from a few well-studied PFAS, the structure-related toxicokinetics of a broader set of PFAS remain unclear. Objectives: To understand the toxicokinetics of PFAS, we attempted to characterize the metabolism pathways of 74 structurally diverse PFAS samples from the U.S. Environmental Protection Agency’s PFAS screening library. Methods: Using the early life stages of zebrafish (Danio rerio) as a model, we determined the bioconcentration factors and phenotypic toxicities of 74 PFAS. Then, we applied high-resolution mass spectrometry–based nontargeted analysis to identify metabolites of PFAS in zebrafish larvae after 5 d of exposure by incorporating retention time and mass spectra. In vitro enzymatic activity experiments with human recombinant liver carboxylesterase (hCES1) were employed to validate the structure-related hydrolysis of 11 selected PFAS. Results: Our findings identified five structural categories of PFAS prone to metabolism. The metabolism pathways of PFAS were highly related to their structures as exemplified by fluorotelomer alcohols that the predominance of β-oxidation or taurine conjugation pathways were primarily determined by the number of hydrocarbons. Hydrolysis was identified as a major metabolism pathway for diverse PFAS, and perfluoroalkyl carboxamides showed the highest in vivo hydrolysis rates, followed by carboxyesters and sulfonamides. The hydrolysis of PFAS was verified with recombinant hCES1, with strong substrate preferences toward perfluoroalkyl carboxamides. Conclusions: We suggest that the roadmap of the structure-related metabolism pathways of PFAS established in this study would provide a starting point to inform the potential health risks of other PFAS. https://doi.org/10.1289/EHP7169

Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA. RNA-seq of neural cells depleted of ARGLU1 revealed significant changes in the expression and alternative splicing of distinct genes involved in neurogenesis. Loss of ARGLU1 is embryonic lethal in mice, and knockdown in zebrafish causes neurodevelopmental and heart defects. Treatment with dexamethasone, a GR activator, also induces changes in the pattern of alternatively spliced genes, many of which were lost when ARGLU1 was absent. Importantly, the genes found to be alternatively spliced in response to glucocorticoid treatment were distinct from those under transcriptional control by GR, suggesting an additional mechanism of glucocorticoid action is present in neural cells. Our results thus show that ARGLU1 is a novel factor for embryonic development that modulates basal transcription and alternative splicing in neural cells with consequences for glucocorticoid signaling.

Brominated azo dyes (BADs) have been identified as predominant indoor brominated pollutants in daycare dust; thus, their potential health risk to children is of concern. However, the toxicities of BADs remain elusive. In this study, the toxicokinetics of two predominant BADs, Disperse Blue 373 (DB373) and Disperse Violet 93 (DV93), and their suspect metabolite 2-bromo-4,6-dinitroaniline (BDNA) was investigated in embryos of zebrafish (

The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated a Tg(QUAS:GFPNLS) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes.

We have investigated the relationship between the function of the gene hindsight (hnt), which is the Drosophila homolog of Ras Responsive Element Binding protein-1 (RREB-1), and the EGFR signaling pathway. We report that hnt mutant embryos are defective in EGFR signaling dependent processes, namely chordotonal organ recruitment and oenocyte specification. We also show the temperature sensitive hypomorphic allele hntpebbled is enhanced by the hypomorphic MAPK allele rolled (rl1). We find that hnt overexpression results in ectopic DPax2 expression within the embryonic peripheral nervous system, and we show that this effect is EGFR-dependent. Finally, we show that the canonical U-shaped embryonic lethal phenotype of hnt, which is associated with premature degeneration of the extraembyonic amnioserosa and a failure in germ band retraction, is rescued by expression of several components of the EGFR signaling pathway (sSpi, Ras85DV12, pntP1) as well as the caspase inhibitor p35. Based on this collection of corroborating evidence, we suggest that an overarching function of hnt involves the positive regulation of EGFR signaling.

The phenomenon of the cytoplasmic localisation of mitochondrial ribosomal subunits (12 S mitochondrial rRNA and 16 S mitochondrial rRNA) has been discovered by scientific teams working with spermatogenic cells of mice. Previous reports showed that the release of mitochondrial substance occurs during interaction of mitochondria with the germ plasm granules (GG). To determine if the interplay between the vasa-positive GG and the mitochondria is associated with cytoplasmic localisation of mtrRNAs, we studied the spermatogenic cells of zebrafish, Danio rerio. It was revealed that in type A undifferentiated spermatogonia the GG did not contact mitochondria, and the extra-mitochondrial localisation of the mtrRNAs was not found. In type A differentiated spermatogonia, the amount of GG in contact with mitochondria increased, but the extra-mitochondrial localisation of the mtrRNAs was not found either. In type B late spermatogonia, which are pre-meiotic cells, the GG/mitochondrion complexes were typically found in contact with the nucleus. This stage was associated with the intra-mitochondrial localisation of GG-originated vasa and extra-mitochondrial localisation of 12 S mtrRNA and 16 S mtrRNA. Until the onset of meiosis, which was determined by the observation of synaptonemal complexes in zygotene-pachytene spermatocytes I, the GG/mitochondrion complexes disappeared, but both types of mtrRNAs persisted in the cytoplasm of spermatids and spermatozoa.

[This corrects the article DOI: 10.1371/journal.ppat.1007597.].

Current peroxisome proliferator-activated receptor (PPAR)-targeted drugs, such as the PPARγ-directed diabetes drug rosiglitazone, are associated with undesirable side effects due to robust agonist activity in non-target tissues. To find new PPAR ligands with fewer toxic effects, we generated transgenic zebrafish that can be screened in high throughput for new tissue-selective PPAR partial agonists. A structural analog of coenzyme Q10 (idebenone) that elicits spatially restricted partial agonist activity for both PPARα and PPARγ was identified. Coenzyme Q10 was also found to bind and activate both PPARs in a similar fashion, suggesting an endogenous role in relaying the states of mitochondria, peroxisomes and cellular redox to the two receptors. Testing idebenone in a mouse model of type 2 diabetes revealed the ability to reverse fatty liver development. These findings indicate new mechanisms of action for both PPARα and PPARγ, and new potential treatment options for nonalcoholic fatty liver disease (NAFLD) and steatosis. This article has an associated First Person interview with the first author of the paper., Summary: A zebrafish screen identifies a novel PPARα/γ ligand, idebenone, with potential for treatment of fatty liver diseases, as seen by testing it in a mouse model of type 2 diabetes.

The observation that long noncoding RNAs (lncRNAs) represent the majority of transcripts in humans has led to a rapid increase in interest and study. Most of this interest has focused on their roles in the nucleus. However, increasing evidence is beginning to reveal even more functions outside the nucleus, and even outside cells. Many of these roles are mediated by newly discovered properties, including the ability of lncRNAs to interact with lipids, membranes, and disordered protein domains, and to form differentially soluble RNA–protein sub-organelles. This review explores the possibilities enabled by these new properties and abilities, such as likely roles in exosome formation and function.

Germline cells segregate from the soma to maintain their totipotency, but the cellular mechanisms of this segregation are unclear. The Drosophila melanogaster embryo forms a posterior group of primordial germline cells (PGCs) by their division from the syncytial soma. Extended plasma membrane furrows enclose the PGCs in response to the germ plasm protein Germ cell-less (Gcl) and Rho1–actomyosin activity. Recently, we found that loss of the Arf-GEF Steppke (Step) leads to similar Rho1-dependent plasma membrane extensions but from pseudocleavage furrows of the soma. Here, we report that the loss of step also leads to premature formation of a large cell group at the anterior pole of the embryo . These anterior cells lacked germ plasm, but budded and formed at the same time as posterior PGCs, and then divided asynchronously as PGCs also do. With genetic analyses we found that Step normally activates Arf small G proteins and antagonizes Rho1–actomyosin pathways to inhibit anterior cell formation. A uniform distribution of step mRNA around the one-cell embryo cortex suggested that Step restricts cell formation through a global control mechanism. Thus, we examined the effect of Step on PGC formation at the posterior pole. Reducing Gcl or Rho1 levels decreased PGC numbers, but additional step RNAi restored their numbers. Reciprocally, GFP–Step overexpression induced dosage- and Arf-GEF-dependent loss of PGCs, an effect worsened by reducing Gcl or actomyosin pathway activity. We propose that a global distribution of Step normally sets an inhibitory threshold for Rho1 activity to restrict early cell formation to the posterior.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

The past decade has seen a major increase in the study of noncoding RNAs (ncRNAs). However, there remains a great deal of confusion and debate over the levels of functionality and mechanisms of action of the majority of these new transcripts. This Opinion article addresses several of these issues, focusing particularly on long ncRNAs (lncRNAs). We reemphasize the unique abilities of RNAs to form myriad structures as well as to interact with other RNAs, DNA, and proteins, which provide them with unique and powerful abilities. One of these, the ability to interact sequence specifically with DNA, has been largely overlooked. Accumulating evidence suggests that evolution has taken advantage of RNA's properties via the rapid acquisition of new noncoding genes in testes, with subsequent gains of function in other tissues. This amplification process appears to be one of the major forces driving metazoan evolution and diversity.

Summary The faithful execution of embryogenesis relies on the ability of organisms to respond to genotoxic stress and to eliminate defective cells that could otherwise compromise viability. In syncytial-stage Drosophila embryos, nuclei with excessive DNA damage undergo programmed elimination through an as-yet poorly understood process of nuclear fallout at the midblastula transition. We show that this involves a Chk2-dependent mechanism of mRNA nuclear retention that is induced by DNA damage and prevents the translation of specific zygotic mRNAs encoding key mitotic, cytoskeletal, and nuclear proteins required to maintain nuclear viability. For histone messages, we show that nuclear retention involves Chk2-mediated inactivation of the Drosophila stem loop binding protein (SLBP), the levels of which are specifically depleted in damaged nuclei following Chk2 phosphorylation, an event that contributes to nuclear fallout. These results reveal a layer of regulation within the DNA damage surveillance systems that safeguard genome integrity in eukaryotes.

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis., Author summary Cryptococcus neoformans is an opportunistic fungal pathogen that is responsible for significant numbers of deaths in the immunocompromised population worldwide. Here we address whether eicosanoids produced by C. neoformans manipulate host innate immune cells during infection. Cryptococcus neoformans produces several eicosanoids that are notable for their similarity to vertebrate eicosanoids, it is therefore possible that fungal-derived eicosanoids may provoke physiological effects in the host. Using a combination of in vitro and in vivo infection models we identify a specific eicosanoid species—prostaglandin E2 –that is required by C. neoformans for growth during infection. We subsequently show that prostaglandin E2 must be converted to 15-keto-prostaglandin E2 within the host before it has these effects. Furthermore, we find that prostaglandin E2/15-keto-prostaglandin E2 mediated virulence is via activation of host PPAR-γ –an intracellular eicosanoid receptor known to interact with 15-keto-PGE2.

Previous work has shown that many of the 18 family members of Drosophila nuclear receptor transcription factors function in a temporal hierarchy to coordinate developmental progression and growth with the rate limiting process of metabolism. To gain further insight into these interactions and processes, we have undertaken a whole-family analysis of nuclear receptor mRNA spatial expression patterns over the entire process of embryogenesis, as well as the 3rd instar wandering larva stage, by using high-resolution fluorescence in situ hybridization. Overall, the patterns of expression are remarkably consistent with previously mapped spatial activity profiles documented during the same time points, with similar hot spots and temporal profiles in endocrine and metabolically important tissues. Among the more remarkable of the findings is that the majority of mRNA expression patterns observed show striking subcellular distributions, indicating potentially critical roles in the control of protein synthesis and subsequent subcellular distributions. These patterns will serve as a useful reference for future studies on the tissue-specific roles and interactions of nuclear receptor proteins, partners, cofactors and ligands.

Regulation of the Hippo pathway via phosphorylation of Yorkie (Yki), the Drosophila homolog of human Yes-associated protein 1, is conserved from Drosophila to humans. Overexpression of a non-phosphorylatable form of Yki induces severe overgrowth in adult fly eyes. Here, we show that yki mRNA associates with microsomal fractions and forms foci that partially colocalize to processing bodies in the vicinity of endoplasmic reticulum. This localization is dependent on a stem-loop (SL) structure in the 3’ untranslated region of yki. Surprisingly, expression of SL deleted yki in eye imaginal discs also results in severe overgrowth phenotypes. When the structure of the SL is disrupted, Yki protein levels increase without a significant effect on RNA levels. When the SL is completely removed, protein levels drastically increase, but in this case, due to increased RNA stability. In the latter case, we show that the increased RNA accumulation is due to removal of a putative miR-8 seed sequence in the SL. These data demonstrate the function of two novel regulatory mechanisms, both controlled by the yki SL element, that are essential for proper Hippo pathway mediated growth regulation.

The Drosophila trithorax- and Polycomb-group (trxG and PcG) proteins maintain activated and repressed transcriptional states at specific target gene loci. The Additional sex combs (Asx) gene is of particular interest as it appears to function in both protein complexes and yet its effects on target genes are more restricted. A novel protein, Tantalus (TAN), was identified in a yeast two-hybrid screen for ASX-interacting proteins that might confer tissue-specific ASX functions. TAN contains consensus nuclear localization sites and binds DNA in vitro. However, its subcellular localization varies in a tissue-specific fashion. In salivary glands, TAN is predominantly nuclear and associates with 66 euchromatic sites on polytene chromosomes, more than half of which overlap with ASX. These loci do not include the homeotic genes of the ANT and BX complexes bound by other PcG and trxG proteins. Rather, tan mutant defects are restricted to sensory organs. We show that one of these defects, shared by Asx, is genetically enhanced by Asx. Taken together, the data suggest that TAN is a tissue-specific cofactor for ASX, and that its activity may be partially controlled by subcellular trafficking.

In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided.

The interaction between the orphan nuclear receptor FTZ-F1 (Fushi tarazu factor 1) and the segmentation gene protein FTZ is critical for specifying alternate parasegments in the Drosophila embryo. Here, we have determined the structure of the FTZ-F1 ligand-binding domain (LBD)·FTZ peptide complex using x-ray crystallography. Strikingly, the ligand-binding pocket of the FTZ-F1 LBD is completely occupied by helix 6 (H6) of the receptor, whereas the cofactor FTZ binds the co-activator cleft site of the FTZ-F1 LBD. Our findings suggest that H6 is essential for transcriptional activity of FTZ-F1; this is further supported by data from mutagenesis and activity assays. These data suggest that FTZ-F1 might belong to a novel class of ligand-independent nuclear receptors. Our findings are intriguing given that the highly homologous human steroidogenic factor-1 and liver receptor homolog-1 LBDs exhibit sizable ligand-binding pockets occupied by putative ligand molecules.

The zebrafish (Danio rerio) is an emerging vertebrate model for drug discovery that permits whole animal drug screens with excellent throughput, combined with ease of use and low cost. This review will begin with a discussion on the background, suitability, and advantages of this vertebrate model system and then, citing specific examples, will describe the utility of zebrafish at specific stages in the drug development pipeline. We will end with a synopsis of recent drug screens based on morphological disruptions, genetic disease models, fluorescent markers, behavioral changes, and specific targets. The numerous advantages of this whole animal approach provide new promise for the discovery of safe, specific, and powerful new drugs.

Nuclear receptors E75, which regulates development in Drosophila melanogaster, and Rev-erbbeta, which regulates circadian rhythm in humans, bind heme within their ligand binding domains (LBD). The heme-bound ligand binding domains of E75 and Rev-erbbeta were studied using electronic absorption, MCD, resonance Raman, and EPR spectroscopies. Both proteins undergo redox-dependent ligand switching and CO- and NO-induced ligand displacement. In the Fe(III) oxidation state, the nuclear receptor hemes are low spin and 6-coordinate with cysteine(thiolate) as one of the two axial heme ligands. The sixth ligand is a neutral donor, presumably histidine. When the heme is reduced to the Fe(II) oxidation state, the cysteine(thiolate) is replaced by a different neutral donor ligand, whose identity is not known. CO binds to the Fe(II) heme in both E75(LBD) and Rev-erbbeta(LBD) opposite a sixth neutral ligand, plausibly the same histidine that served as the sixth ligand in the Fe(III) state. NO binds to the heme of both proteins; however, the NO-heme is 5-coordinate in E75 and 6-coordinate in Rev-erbbeta. These nuclear receptors exhibit coordination characteristics that are similar to other known redox and gas sensors, suggesting that E75 and Rev-erbbeta may function in heme-, redox-, or gas-regulated control of cellular function.

Genome expression profiling has led to the important realization that RNA molecules are more numerous and diverse than previously expected. One aspect of RNA biology that is just beginning to be fully appreciated is the extent to which mRNAs are transported to specific subcellular destinations before being translated, an exquisite mechanism for targeting proteins where they are required in the cell. While several excellent reviews have discussed the subject of mRNA localization, it is only in recent years that high-throughput technologies have been applied to address issues such as the extent and diversity of RNA localization events and mechanisms. This review focuses on these recent functional genomic approaches, their implications, and the new tools and methods that will be needed to further elucidate mRNA localization pathways.

Synaptic transmission is highly plastic and subject to regulation by a wide variety of neuromodulators and neuropeptides. In the present study, we have examined the role of isoforms of the cytochrome b561 homologue called no extended memory (nemy) in regulation of synaptic strength and plasticity at the neuromuscular junction (NMJ) of third instar larvae in Drosophila. Specifically, we generated two independent excisions of nemy that differentially affect the expression of nemy isoforms. We show that the nemy 45 excision, which specifically reduces the expression of the longest splice form of nemy, leads to an increase in stimulus evoked transmitter release and altered synaptic plasticity at the NMJ. Conversely, the nemy 26.2 excision, which appears to reduce the expression of all splice forms except the longest splice isoform, shows a reduction in stimulus evoked transmitter release, and enhanced synaptic plasticity. We further show that nemy 45 mutants have reduced levels of amidated peptides similar to that observed in peptidyl-glycine hydryoxylating mono-oxygenase (PHM) mutants. In contrast, nemy 26.2 mutants show no defects in peptide amidation but rather display a decrease in Tyramine β hydroxylase activity (TβH). Taken together, these results show non-redundant roles for the different nemy isoforms and shed light on the complex regulation of neuromodulators.

SummaryNuclear 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.

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins.

The homeodomain protein Fushi tarazu (Ftz) is required for several embryonic patterning processes including segmentation and neurogenesis. During the stages that these processes are regulated the protein is differentially phosphorylated, suggesting that phosphorylation plays a role in helping the protein to regulate different functions in different tissues. We showed in a recent study that one of the Ftz phosphorylation sites, a protein kinase A-type site in the N-terminal arm of the homeodomain, is required for normal Ftz-dependent segmentation. Here we test whether phosphorylation of this site (Thr-263) is also required in the developing central nervous system (CNS). A well-established role for Ftz in the CNS is for the differentiation of neurons referred to as RP2 neurons. Absence of Ftz expression in these cells causes a failure of certain target genes to be expressed and subsequent defects in RP2 differentiation. In contrast to its effect on segmentation, we find that mutation of Thr-263 to Ala (or Asp) has no effect on these CNS functions. This suggests that the phosphorylation state of this site is irrelevant for Ftz function in the CNS, and that there are tissue-specific differences in the requirements for Ftz phosphorylation.

Scalloped (Sd) and Vestigial (Vg) are each needed forDrosophila 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 tissue-specific transcriptional intermediary factor of Sd.

The Drosophila homeodomain-containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage-specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue-specific Ftz activities. Here we show that one of the Ftz phosphorylation sites, T263 in the N-terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non-phosphorylatable residue Ala resulted in loss of ftz-dependent segments. Conversely, substitution of T263 with Asp, which is also non-phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA-binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain-mediated interaction with an embryonically expressed protein.

Aven is a regulator of the DNA damage response and G2/M cell cycle progression. Overexpression of Aven is associated with poor prognosis in patients with childhood acute lymphoblastic leukemia and acute myeloid leukemia, and altered intracellular Aven distribution is associated with infiltrating ductal carcinoma and papillary carcinoma breast cancer subtypes. Although Aven orthologs have been identified in most vertebrate species, no Aven gene has been reported in invertebrates. Here, we describe a Drosophila melanogaster open reading frame (ORF) that shares sequence and functional similarities with vertebrate Aven genes. The protein encoded by this ORF, which we named dAven, contains several domains that are highly conserved among Aven proteins of fish, amphibian, bird and mammalian origins. In flies, knockdown of dAven by RNA interference (RNAi) resulted in lethality when its expression was reduced either ubiquitously or in fat cells using Gal4 drivers. Animals undergoing moderate dAven knockdown in the fat body had smaller fat cells displaying condensed chromosomes and increased levels of the mitotic marker phosphorylated histone H3 (PHH3), suggesting that dAven was required for normal cell cycle progression in this tissue. Remarkably, expression of dAven in Xenopus egg extracts resulted in G2/M arrest that was comparable to that caused by human Aven. Taken together, these results suggest that, like its vertebrate counterparts, dAven plays a role in cell cycle regulation. Drosophila could be an excellent model for studying the function of Aven and identifying cellular factors that influence its activity, revealing information that may be relevant to human disease.

During Drosophila development, an important aspect of body patterning is the division of the embryo into repeating morphological units referred to as parasegments. The parasegmental domains are first defined at the blastoderm stage by alternating stripes of transcripts encoded by the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) and later by stripes encoded by the segment polarity genes engrailed (en) and wingless. Here, we show that the runt gene (run) is required to generate asymmetries within these parasegmental domains. Using a heat-shock-inducible run transgene, we found that ectopic run expression leads to rapid repression of eve stripes and a somewhat delayed expansion of ftz stripes. Unexpectedly, we also found that ectopic run was a rapid and potent repressor of odd-numbered en stripes. Two remarkably different segmental phenotypes were generated as a consequence of these effects. In solving the mechanisms underlying these phenotypes, we discovered that the positioning of en stripes is largely determined by the actions of negative regulators. Our data indicate that run is required to limit the domains of en expression in the odd-numbered parasegments, while the odd-skipped gene is required to limit the domains of en expression in the even-numbered parasegments. Activation of en at the anterior margins of both sets of parasegments requires the repression of run and odd by the product of the eve gene. The spatial restriction of gene expression via negative and double negative pathways such as these is likely to be a common theme during development.

The Drosophila pair-rule gene even-skipped (eve) encodes a homeo-domain-containing protein (Eve) that is required for the development of both odd- and even-numbered parasegments. We have used a heat shock-inducible eve transgene to study the regulatory functions of Eve in vivo. Transcripts encoded by eight other segmentation genes were monitored for changes in distribution and abundance following short pulses of ectopic Eve expression. Two tiers of response times appeared to distinguish between genes that were direct [fushi tarazu (ftz), odd-skipped (odd), runt (run), paired, and wingless] and indirect [eve, hairy, and engrailed (en)] targets of Eve. Genes that appeared to be directly regulated by Eve were differentially repressed in a concentration-dependent fashion. Interestingly, the run and ftz genes could also be activated by Eve during a brief 20- to 30-min stage in development. The delayed actions upon the eve and en genes appeared to be mediated by run and odd. As in eve- embryos, these effects on segmentation gene expression patterns caused defects in both odd- and even-numbered parasegments. Four sequential phenotypes could be induced, each of which was attributable to the altered expression of a unique subset of target genes.