The Drosophila ortholog of the human XPG gene.

Xeroderma pigmentosum complementation group G (XPG) protein is a junction-specific endonuclease which is indispensable for nucleotide excision repair (NER) of DNA in eukaryotes. Recent studies have hinted at a second, essential function for the XPG protein in higher eukaryotes. We undertook a comparison of the amino acid sequences of multiple XPG orthologs to determine if a motif or domain could be identified that is conserved uniquely in higher eukaryotes. A search of current databases allowed us to retrieve complete amino acid sequences for the human, mouse and Xenopus XPG proteins, and for two yeast orthologs. We also identified an incomplete Drosophila open reading frame (ORF) that was a good candidate for the XPG protein. We cloned a complete Drosophila cDNA for this ORF and examination of the primary amino acid sequence suggests that this cDNA encodes the Drosophila ortholog of XPG. A comparison of all six orthologous polypeptides reveals the presence of two previously unidentified conserved domains. One of these is unique to all four higher eukaryotic sequences. Conceivably this domain evolved to support the essential function of XPG protein.     

The acquisition of resistance to ecdysteroids in cultured Drosophila cells

Drosophila melanogaster K_c cells become refractory toward ecdysteroids after 4 days of exposure to the molting hormone, 20-OH-ecdysone. Associated with the appearance of hormonal insensitivity is a loss of ecdysteroid receptors. Hormone-resistant cells maintain a low level of receptor that is indistinguishable from that of responsive, hormonally naive cells. After extended periods in culture, ecdysteroid receptor content in previously exposed cells returns to that of naive control cells. The reappearance of receptor is coincident with the resumption of hormonally induced growth inhibition.     

The response of Drosophila imaginal disc cell lines to ecdysteroids

Summary We have investigated the action of the moulting hormone 20-hydroxy ecdysone (20-HOE) on our leg and wing imaginal disc cell lines. At the morphological level, cells stop dividing and there is some cell death. The remaining cells elongate and aggregate, often producing long processes which form connections between different aggregates. 20-HOE acts within the first one or two days of a passage, at an optimum concentration of 10 ng/ml, this being about 1/100 of the optimum for ecdysone. One cloned wing cell line, C9, has been found to be relatively insensitive to the action of 20-HOE. We have been able to select for resistance to 20-HOE by growing cells in gradually increasing concentrations of hormone followed by passages in hormone-free medium. This has enabled us to isolate a wing cell line C1.8R from its parent cloned line C1.8+. This shows no response to 20-HOE, and cell growth continues even at hormone concentrations as high as 150 ng/ml. We have measured chitin synthesis by the incorporation of radioactive glucosamine into a cell fraction resistant to extensive alkali hydrolysis. The residue was incubated with chitinase, which resulted in a 50% reduction in labelled product. Treatment with 10 ng/ml of 20-HOE dramatically increased chitin synthesis in line C1.8+, but had no effect in the line C1.8R, selected for resistance to hormone. Correspondence to: M.J. Milner     

Temporal regulation of metamorphic processes in Drosophila by the let-7 and miR-125 heterochronic microRNAs

BACKGROUND: The let-7 and lin-4 microRNAs belong to a class of temporally expressed, noncoding regulatory RNAs that function as heterochronic switch genes in the nematode C. elegans. Heterochronic genes control the relative timing of events during development and are considered a major force in the rapid evolution of new morphologies. let-7 is highly conserved and in Drosophila is temporally coregulated with the lin-4 homolog, miR-125. Little is known, however, about their requirement outside the nematode or whether they universally control the timing of developmental processes. RESULTS: We report the generation of a Drosophila mutant that lacks let-7 and miR-125 activities and that leads to a pleiotropic phenotype arising during metamorphosis. We focus on two defects and demonstrate that loss of let-7 and miR-125 results in temporal delays in two distinct metamorphic processes: the terminal cell-cycle exit in the wing and maturation of neuromuscular junctions (NMJs) at adult abdominal muscles. We identify the abrupt (ab) gene, encoding a nuclear protein, as a bona fide let-7 target and provide evidence that let-7 governs the maturation rate of abdominal NMJs during metamorphosis by regulating ab expression. CONCLUSIONS: Drosophila Iet-7 and miR-125 mutants exhibit temporal misregulation of specific metamorphic processes. As in C. elegans, Drosophila let-7 is both necessary and sufficient for the appropriate timing of a specific cell-cycle exit, indicating that its function as a heterochronic microRNA is conserved. The ab gene is a target of let-7, and its repression in muscle is essential for the timing of NMJ maturation during metamorphosis. Our results suggest that let-7 and miR-125 serve as conserved regulators of events necessary for the transition from juvenile to adult life stages.     

Analyzing the repressive function of ultraspiracle, the Drosophila RXR, in Drosophila eye development.

Response to the insect hormone ecdysone is mediated by a nuclear receptor complex containing Ultraspiracle (USP) and the Ecdysone Receptor (EcR). Among other phenotypes, loss of functional USP in Drosophila eye development results in an accelerated morphogenetic furrow, although loss of ecdysone arrests the furrow. We have shown that USP both represses and activates a gene affecting furrow movement, the ecdysone-responsive Z1 isoform of Broad-Complex, and we report additional usp mutant phenotypes. Using targeted replacement of USP to rescue usp mutant clones in the eye, we have mapped various USP functions and tested whether the USP nuclear receptor has an activating as well as a repressive effect on furrow movement. Furrow movement and related phenotypes are rescued by the presence of USP in a limited domain near the furrow while other phenotypes are rescued by USP expression posterior to the furrow. Our data indicate roles for USP activity at multiple developmental stages and help explain why loss of functional USP leads to furrow advancement while loss of ecdysone stops furrow movement.     

The Drosophila ortholog of the endolysosomal membrane protein, endolyn, regulates cell proliferation

Endolyn (CD164) is a sialomucin that regulates the proliferation, adhesion, and migration of human haematopoietic stem and progenitor cells. This molecule is predominately localized in endocytotic compartments, where it may contribute to endolysosomal biogenesis and trafficking. In order to more closely define the function of endolyn from an evolutionary view-point, we first analyzed endolyn orthologs in species ranging from insects, fish, and birds to mammals. The predicted molecular structures of the endolyn orthologs from these species are well conserved, particularly with respect to significant O-linked glycosylation of the extracellular domain, and the high degree of amino acid similarities within their transmembrane and cytoplasmic domains, with the latter possessing the lysosomal target signal, YXXphi. Focusing on Drosophila, our studies showed that the subcellular distribution of endolyn in non-polarized Drosophila S2 cells resembles that of its human counterpart in hematopoietic cells, with its predominant localization being within intracellular vesicles, while a small fraction occurs on the cell surface. Both Y --> A and L --> A mutations in the YHTL motif perturbed the normal subcellular distribution of Drosophila endolyn. Interestingly, embryonic and early larval development was often arrested in endolyn-deficient Drosophila mutants. This may partly be due to the role of endolyn in regulating cell proliferation, since knock-down of endolyn expression in S2 cells resulted in up to 50% inhibition of cell growth, with a proportion of cells undergoing apoptosis. Taken together, these results demonstrate that endolyn is an evolutionarily conserved sialomucin fundamentally involved in cell proliferation in both the human and Drosophila melanogaster.     

Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.     

The Drosophila ortholog of the human Wnt inhibitor factor Shifted controls the diffusion of lipid-modified Hedgehog

The Hedgehog (Hh) family of morphogenetic proteins has important instructional roles in metazoan development and human diseases. Lipid modified Hh is able to migrate to and program cells far away from its site of production despite being associated with membranes. To investigate the Hh spreading mechanism, we characterized Shifted (Shf) as a component in the Drosophila Hh pathway. We show that Shf is the ortholog of the human Wnt inhibitory factor (WIF), a secreted antagonist of the Wingless pathway. In contrast, Shf is required for Hh stability and for lipid-modified Hh diffusion. Shf colocalizes with Hh in the extracellular matrix and interacts with the heparan sulfate proteoglycans (HSPG), leading us to suggest that Shf could provide HSPG specificity for Hh. We also show that human WIF inhibits Wg signaling in Drosophila without affecting the Hh pathway, indicating that different WIF family members might have divergent functions in each pathway.     

Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects

The major postembryonic developmental events happening in insect life, including molting and metamorphosis, are regulated and coordinated temporally by pulses of ecdysone. The biological activity of this steroid hormone is mediated by two nuclear receptors: the ecdysone receptor (EcR) and the Ultraspiracle protein (USP). The crystal structure of the ligand-binding domain from the lepidopteran Heliothis virescens USP reported here shows that the loop connecting helices H1 and H3 precludes the canonical agonist conformation. The key residues that stabilize this unique loop conformation are strictly conserved within the lepidopteran USP family. The presence of an unexpected bound ligand that drives an unusual antagonist conformation confirms the induced-fit mechanism accompanying the ligand binding. The ligand-binding pocket exhibits a retinoid X receptor-like anchoring part near a conserved arginine, which could interact with a USP ligand functional group. The structure of this receptor provides the template for designing inhibitors, which could be utilized as a novel type of environmentally safe insecticides.     

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We demonstrate a role for the p21(CIP)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. Our data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts.     

The Drosophila Polycomb group gene Sex combs extra encodes the ortholog of mammalian Ring1 proteins

In Drosophila, the Polycomb group (PcG) of genes is required for the maintenance of homeotic gene repression during development. Here, we have characterized the Drosophila ortholog of the products of the mammalian Ring1/Ring1A and Rnf2/Ring1B genes. We show that Drosophila Ring corresponds to the Sex combs extra (Sce), a previously described PcG gene. We find that Ring/Sce is expressed and required throughout development and that the extreme Pc embryonic phenotype due to the lack of maternal and zygotic Sce can be rescued by ectopic expression of Ring/Sce. This phenotypic rescue is also obtained by ectopic expression of the murine Ring1/Ring1A, suggesting a functional conservation of the proteins during evolution. In addition, we find that Ring/Sce binds to about 100 sites on polytene chromosomes, 70% of which overlap those of other PcG products such as Polycomb, Posterior sex combs and Polyhomeotic, and 30% of which are unique. We also show that Ring/Sce interacts directly with PcG proteins, as it occurs in mammals.     

Drosophila wing development in the absence of dorsal identity

The developing wing disc of Drosophila is divided into distinct lineage-restricted compartments along both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes. At compartment boundaries, morphogenic signals pattern the disc epithelium and direct appropriate outgrowth and differentiation of adult wing structures. The mechanisms by which affinity boundaries are established and maintained, however, are not completely understood. Compartment-specific adhesive differences and inter-compartment signaling have both been implicated in this process. The selector gene apterous (ap) is expressed in dorsal cells of the wing disc and is essential for D/V compartmentalization, wing margin formation, wing outgrowth and dorsal-specific wing structures. To better understand the mechanisms of Ap function and compartment formation, we have rescued aspects of the ap mutant phenotype with genes known to be downstream of Ap. We show that Fringe (Fng), a secreted protein involved in modulation of Notch signaling, is sufficient to rescue D/V compartmentalization, margin formation and wing outgrowth when appropriately expressed in an ap mutant background. When Fng and alphaPS1, a dorsally expressed integrin subunit, are co-expressed, a nearly normal-looking wing is generated. However, these wings are entirely of ventral identity. Our results demonstrate that a number of wing development features, including D/V compartmentalization and wing vein formation, can occur independently of dorsal identity and that inter-compartmental signaling, refined by Fng, plays the crucial role in maintaining the D/V affinity boundary. In addition, it is clear that key functions of the ap selector gene are mediated by only a small number of downstream effectors.     

RXR receptor agonist suppression of thyroid function: central effects in the absence of thyroid hormone receptor

High-affinity agonists for the retinoic acid X receptors (RXR) have pleotropic effects when administered to humans. These include induction of hypertriglyceridemia and hypothyroidism. We determined the effect of a novel high-affinity RXR agonist with potent antihyperglycemic effects on thyroid function of female Zucker diabetic rats and nondiabetic littermates and in db/db mice. In both nondiabetic and ZFF rats, AGN194204 causes a 70-80% decrease in thyrotropin (TSH), 3,3',5-triiodothyronine, and thyroxine (T(4)) concentrations. In the db/db mouse, AGN194204 causes a time-dependent decrease in thyroid hormone levels with the fall in TSH that was significant after 1 day of treatment preceding the fall in T(4) levels that was significant at 3 days of treatment. Treatment with AGN194204 caused an initial increase in hepatic 5'-deiodinase mRNA levels which then fell to undetectable levels by 3 days of treatment and continued to be low at 7 days of treatment. After treatment for 5 days with AGN194204, both wild-type and thyroid hormone receptor beta (TR beta(-/-))-deficient mice demonstrated a nearly 50% decrease in serum TSH and T(4) concentrations. The results suggest that a high-affinity RXR agonist with antihyperglycemic activity can cause central hypothyroidism independently of TR beta, the main mediator of hormone-induced TSH suppression.     

Identification of dAven,a Drosophila melanogaster ortholog of the cell cycle regulator Aven: Identification of Drosophila Aven

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.Key words: Drosophila melanogaster, Aven, Ataxia telangiectasia mutated, ATM and Rad 3-related, cell cycle, checkpoint