Evolution and functional characterisation of the IR chemosensory receptors in the Drosophila larval gustatory system

Chemosensory receptor gene families encode divergent proteins capable of detecting a huge diversity of environmental stimuli that are constantly changing over evolutionary time as organisms adapt to distinct ecological niches. While olfaction is dedicated to the detection of volatile compounds, taste is key to assess food quality for nutritional value and presence of toxic substances. The sense of taste also provides initial signals to mediate endocrine regulation of appetite and food metabolism and plays a role in kin recognition. The fruit fly Drosophila melanogaster is a very good model for studying smell and taste because these senses are very important in insects and because a broad variety of genetic tools are available in Drosophila. Recently, a family of 66 chemosensory receptors, the Ionotropic Receptors (IRs) was described in fruit flies. IRs are distantly related to ionotropic glutamate receptors (iGluRs), but their evolutionary origin from these synaptic receptors is unclear. While 16 IRs are expressed in the olfactory system, nothing is known about the other members of this repertoire. In this thesis, I describe bioinformatic, expression and functional analyses of the IRs aimed at understanding how these receptors have evolved, and at characterising the role of the non-olfactory IRs. I show that these have emerged at the basis of the protostome lineage and probably have acquired their sensory function very early. Moreover, although several IRs are conserved across insects, there are rapid and dramatic changes in the size and divergence of IR repertoires across species. I then performed a comprehensive analysis of IR expression in the larva of Drosophila melanogaster, which is a good model to study taste and feeding mechanisms as it spends most of its time eating or foraging. I found that most of the divergent members of the IR repertoire are expressed in both peripheral and internal gustatory neurons, suggesting that these are involved in taste perception. Finally, through the establishment of a new neurophysiological assay in larvae, I identified for the first time subsets of IR neurons that preferentially detect sugars and amino acids, indicating that IRs might be involved in sensing these compounds. Together, my results indicate that IRs are an evolutionarily dynamic and functionally versatile family of receptors. In contrast to the olfactory IRs that are well-conserved, gustatory IRs are rapidly evolving species-specific receptors that are likely to be involved in detecting a wide variety of tastants. - La plupart des animaux possèdent de grandes familles de récepteurs chimiosensoriels dont la fonction est de détecter l'immense diversité de composés chimiques présents dans l'environnement. Ces récepteurs évoluent en même temps que les organismes s'adaptent à leur écosystème. Il existe deux manières de percevoir ces signaux chimiques : l'olfaction et le goût. Alors que le système olfactif perçoit les composés volatiles, le sens du goût permet d'évaluer, par contact, la qualité de la nourriture, de détecter des substances toxiques et de réguler l'appétit et le métabolisme. L'un des organismes modèles les plus pertinents pour étudier le sens du goût est le stade larvaire de la mouche du vinaigre Drosophila melanogaster. En effet, la principale fonction du stade larvaire est de trouver de la nourriture et de manger. De plus, il est possible d'utiliser tous les outils génétiques développés chez la drosophile. Récemment, une nouvelle famille de 66 récepteurs chimiosensoriels appelés Récepteurs Ionotropiques (IRs) a été découverte chez la drosophile. Bien que leur orogine soit peu claire, ces récepteurs sont similaires aux récepteurs ionotropiques glutamatergiques impliqués dans la transmission synaptique. 16 IRs sont exprimés dans le système olfactif de la mouche adulte, mais pour l'instant on ne connaît rien des autres membres de cette famille. Durant ma thèse, j'ai effectué des recherches sur l'évolution de ces récepteurs ainsi que sur l'expression et la fonction des IRs non olfactifs. Je démontre que les IRs sont apparus chez l'ancêtre commun des protostomiens et ont probablement acquis leur fonction sensorielle très rapidement. De plus, bien qu'un certain nombre d'IRs olfactifs soient conservés chez les insectes, d'importantes variations dans la taille et la divergence des répertoires d'IRs entre les espèces ont été constatées. J'ai également découvert qu'un grand nombre d'IRs non olfactifs sont exprimés dans différents organes gustatifs, ce qui leur confère probablement une fonction dans la perception des goûts. Finalement, pour la première fois, des neurones exprimant des IRs ont été identifiés pour leur fonction dans la perception de sucres et d'acides aminés chez la larve. Mes résultats présentent les IRs comme une famille très dynamique, aux fonctions très variées, qui joue un rôle tant dans l'odorat que dans le goût, et dont la fonction est restée importante tout au long de l'évolution. De plus, l'identification de neurones spécialisés dans la perception de certains composés permettra l'étude des circuits neuronaux impliqués dans le traitement de ces informations.

Premenopausal serum androgens and breast cancer risk: a nested case-control study.

ABSTRACT: INTRODUCTION: Prospective epidemiologic studies have consistently shown that levels of circulating androgens in postmenopausal women are positively associated with breast cancer risk. However, data in premenopausal women are limited. METHODS: A case-control study nested within the New York University Women's Health Study was conducted. A total of 356 cases (276 invasive and 80 in situ) and 683 individually-matched controls were included. Matching variables included age and date, phase, and day of menstrual cycle at blood donation. Testosterone, androstenedione, dehydroandrosterone sulfate (DHEAS) and sex hormone-binding globulin (SHBG) were measured using direct immunoassays. Free testosterone was calculated. RESULTS: Premenopausal serum testosterone and free testosterone concentrations were positively associated with breast cancer risk. In models adjusted for known risk factors of breast cancer, the odds ratios for increasing quintiles of testosterone were 1.0 (reference), 1.5 (95% confidence interval (CI), 0.9 to 2.3), 1.2 (95% CI, 0.7 to 1.9), 1.4 (95% CI, 0.9 to 2.3) and 1.8 (95% CI, 1.1 to 2.9; Ptrend = 0.04), and for free testosterone were 1.0 (reference), 1.2 (95% CI, 0.7 to 1.8), 1.5 (95% CI, 0.9 to 2.3), 1.5 (95% CI, 0.9 to 2.3), and 1.8 (95% CI, 1.1 to 2.8, Ptrend = 0.01). A marginally significant positive association was observed with androstenedione (P = 0.07), but no association with DHEAS or SHBG. Results were consistent in analyses stratified by tumor type (invasive, in situ), estrogen receptor status, age at blood donation, and menopausal status at diagnosis. Intra-class correlation coefficients for samples collected from 0.8 to 5.3 years apart (median 2 years) in 138 cases and 268 controls were greater than 0.7 for all biomarkers except for androstenedione (0.57 in controls). CONCLUSIONS: Premenopausal concentrations of testosterone and free testosterone are associated with breast cancer risk. Testosterone and free testosterone measurements are also highly reliable (that is, a single measurement is reflective of a woman's average level over time). Results from other prospective studies are consistent with our results. The impact of including testosterone or free testosterone in breast cancer risk prediction models for women between the ages of 40 and 50 years should be assessed. Improving risk prediction models for this age group could help decision making regarding both screening and chemoprevention of breast cancer.

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells.

T cells move randomly ("random-walk"), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator-activated receptor gamma coactivator 1beta [PGC-1beta], estrogen-related receptor alpha [ERRalpha], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.

Role of amphiregulin in mammary gland development

Abstract Ovarian hormones are key regulators of postnatal mammary gland development and are linked to breast carcinogenesis. In particular, estrogens induce mammary epithelial cells to proliferate at the onset of puberty, leading to the elongation of the rudimental ductal tree into the fatty stromal tissue. Elucidating the molecular events underlying estrogen mitogenic activity in the mammary gland is of value in understanding how the deregulation of this signalling pathway can lead to breast tumorigenesis. Our lab has recently shown that estrogen induces mammary proliferation via epithelial estrogen receptor alpha (ERα) by a paracrine mechanism. Based on the finding that several EGF receptor (EGFR) ligands are able to substitute for estrogens and that amphiregulin (Areg), one of these ligands, is required during mammary development, we have hypothesized that Areg is a key mediator of estrogen induced paracrine signalling during ductal morphogenesis. Our analysis of the Areg -/- mice mammary phenotype reveals that epithelial Areg is required at the onset of puberty for epithelial proliferation, terminal end bud (TEB) formation and, subsequently, ductal elongation. Hormonal stimulation experiments show that among the EGFR ligands, only Arég is specifically controlled by estrogen at the transcriptional level, via ERα, in the mammary gland. Moreover, Areg is required for the estrogen-induced mammotrophic effects of epithelial proliferation and ductal elongation. We have shown that ectopic Areg expression in ERα -/- mammary epithelial cells is sufficient to induce ductal morphogenesis. Our transplantations experiment show that when Areg -/- cells are in the presence of wt cells they contribute to all aspects of ductal development, suggesting that this growth factor acts in a paracrine fashion. Moreover, this result shows that Areg -/- epithelial cells are not intrinsically impaired in proliferation. Our transplantation experiment carried out under physiological conditions confirmed previous reports showing that stromal EGFR is needed for ductal morphogenesis. This suggests that estrogen-driven Areg signalling involves an epithelium-stroma crosstalk. Thus, these data confirmed our hypothesis of Areg being an important estrogen mediator during ductal morphogenesis. Résumé Les hormones ovariennes, régulatrices clés du développement post-natal de la glande mammaire, sont également liées à la carcinogénèse du sein. En particulier, l'oestrogène induit la division des cellules épithéliales au début de la puberté. Cette prolifération amène à l'élongation du réseau canalaire rudimentaire et permet l'invasion du compartiment stromal. L'élucidation des mécanismes moléculaires responsables de l'activité mitogénique de l'oestrogène dans la glande mammaire est précieuse pour une meilleure compréhension du développement du cancer du sein. Notre laboratoire a récemment démontré que l'cestrogène induit la prolifération des cellules épithéliales par un signal paracrine, grâce au récepteur à l'oestrogène alpha (ERα). En se basant sur le fait que plusieurs ligands du récepteur à l'EGF (EGFR) sont capables de se substituer à l'cestrogène et d'induire la prolifération épithéliale et qu'amphiregulin (Areg), un de ces ligands, est essentielle au développement de la glande mammaire, nous avons émi l'hypothèse que Areg est un médiateur essentiel du signal paracrine induit par l'oestrogène pendant la croissance du système canalaire. Nos analyses phénotypiques des glandes mammaires issues de souris transgéniques Areg -/- démontrent que cette protéine est indispensable à la prolifération des cellules épithéliales mammaires au début de la puberté et à la formation des bourgeons terminaux qui conduisent à l'élongation des canaux. Nos expériences de stimulations hormonales démontrent que, parmi l'ensemble des ligands du EGFR, seule Areg est contrôlée au niveau transcriptionnel par l'cestrogène dans la glande mammaire, ceci via le récepteur ERα. De plus, Areg est essentielle pour le effets mammotrophique induit par l'cestrogène, à savoir la prolifération épithéliál et la croissance du système canalaire. Par ailleurs, l'expression ectopique d'Areg dans des cellules epithéliales mammaires de souris transgéniques ERα -/- est suffisante pour permettre la formation du réseau canalaire. En présence de cellules normales, les cellules dépourvues du gène d'Areg contribuent à la formation des canaux. Cette expérience suggère que ce facteur de croissance agit de manière paracrine. De plus, ce résultat montre que les cellules épithéliales Areg -/- conservent leur potentiel prolifératif. Nos expériences de transplantation, réalisées dans des conditions physiologiques, ont confirmé des précédentes études qui montraient que le récepteur stromal à l'EGF est nécessaire pour la morphogénèse du système canalaire. Ceci suggère que la voie de signalisation activée par l'oestrogène et dépendante d' implique une communication entre l'épithélium et le stroma. Ainsi, ces résultats valident notre hypothèse puisqu'ils confirment Areg en tant que médiateur majeur de l'oestrogène dans la morphogénèse du système canalaire.

A Preclinical Model for ERα-Positive Breast Cancer Points to the Epithelial Microenvironment as Determinant of Luminal Phenotype and Hormone Response.

Seventy-five percent of breast cancers are estrogen receptor α positive (ER(+)). Research on these tumors is hampered by lack of adequate in vivo models; cell line xenografts require non-physiological hormone supplements, and patient-derived xenografts (PDXs) are hard to establish. We show that the traditional grafting of ER(+) tumor cells into mammary fat pads induces TGFβ/SLUG signaling and basal differentiation when they require low SLUG levels to grow in vivo. Grafting into the milk ducts suppresses SLUG; ER(+) tumor cells develop, like their clinical counterparts, in the presence of physiological hormone levels. Intraductal ER(+) PDXs are retransplantable, predictive, and appear genomically stable. The model provides opportunities for translational research and the study of physiologically relevant hormone action in breast carcinogenesis.

Gene transfer into Xenopus hepatocytes: transcriptional regulation by members of the nuclear receptor superfamily.

A procedure to culture Xenopus laevis hepatocytes that allows the cells in primary culture to be subjected to gene transfer experiments has been developed. The cultured cells continue to present tissue-specific markers such as expression of the albumin gene or estrogen-controlled vitellogenin gene expression, which are both restricted to liver. Two efficient and reproducible gene transfer procedures have been adapted to the Xenopus hepatocytes, namely lipofection and calcium phosphate-mediated precipitation. The transcription of transfected reporter genes controlled by estrogen-, glucocorticoid- or peroxisome proliferator-response elements was stimulated by endogenous or co-transfected receptor in a ligand-dependent manner. Furthermore, the expression of a reporter gene under the control of the entire promoter of the vitellogenin B1 gene mimicked the expression of the chromosomal vitellogenin gene with respect to basal and estrogen-induced activity. Thus, this culture-transfection system will prove very useful to study the regulation of genes expressed in the liver under the control of various hormones or xenobiotics.

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

Sumoylated PPARalpha mediates sex-specific gene repression and protects the liver from estrogen-induced toxicity in mice.

As most metabolic studies are conducted in male animals, understanding the sex specificity of the underlying molecular pathways has been broadly neglected; for example, whether PPARs elicit sex-dependent responses has not been determined. Here we show that in mice, PPARalpha has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and immunity. In male mice, this effect was reproduced by the administration of a synthetic PPARalpha ligand. Using the steroid oxysterol 7alpha-hydroxylase cytochrome P4507b1 (Cyp7b1) gene as a model, we elucidated the molecular mechanism of this sex-specific PPARalpha-dependent repression. Initial sumoylation of the ligand-binding domain of PPARalpha triggered the interaction of PPARalpha with GA-binding protein alpha (GABPalpha) bound to the target Cyp7b1 promoter. Histone deacetylase and DNA and histone methylases were then recruited, and the adjacent Sp1-binding site and histones were methylated. These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1. Physiologically, this repression conferred on female mice protection against estrogen-induced intrahepatic cholestasis, the most common hepatic disease during pregnancy, suggesting a therapeutic target for prevention of this disease.

Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids.

Hereditary diffuse leukoencephalopathy with spheroids (HDLS) is an autosomal-dominant central nervous system white-matter disease with variable clinical presentations, including personality and behavioral changes, dementia, depression, parkinsonism, seizures and other phenotypes. We combined genome-wide linkage analysis with exome sequencing and identified 14 different mutations affecting the tyrosine kinase domain of the colony stimulating factor 1 receptor (encoded by CSF1R) in 14 families with HDLS. In one kindred, we confirmed the de novo occurrence of the mutation. Follow-up sequencing identified an additional CSF1R mutation in an individual diagnosed with corticobasal syndrome. In vitro, CSF-1 stimulation resulted in rapid autophosphorylation of selected tyrosine residues in the kinase domain of wild-type but not mutant CSF1R, suggesting that HDLS may result from partial loss of CSF1R function. As CSF1R is a crucial mediator of microglial proliferation and differentiation in the brain, our findings suggest an important role for microglial dysfunction in HDLS pathogenesis.

Identification of two steroid-responsive promoters of different strength controlled by the same estrogen-responsive element in the 5'-end region of the Xenopus laevis vitellogenin gene A1.

A structural and functional analysis of the 5'-end region of the Xenopus laevis vitellogenin gene A1 revealed two transcription initiation sites located 1.8 kilobases apart. A RNA polymerase II binding assay indicates that both promoters form initiation complexes efficiently. In vitro, using a transcription assay derived from a HeLa whole-cell extract, the upstream promoter is more than 10-fold stronger than the downstream one. In contrast, both promoters have a similar strength in a HeLa nuclear extract. In vivo, that is in estrogen-stimulated hepatocytes, it is the downstream promoter homologous to the one used by the other members of the vitellogenin gene family, which is 50-fold stronger than the upstream promoter. Thus, if functional vitellogenin mRNA results from this latter activity, it would contribute less than 1% to the synthesis of vitellogenin by fully induced Xenopus hepatocytes expressing the four vitellogenin genes. In contrast, both gene A1 promoters are silent in uninduced hepatocytes. Transfection experiments using the Xenopus cell line B3.2 in which estrogen-responsiveness has been introduced reveal that the strong downstream promoter is controlled by an estrogen responsive element (ERE) located 330 bp upstream of it. The upstream promoter can also be controlled by the same ERE. Since the region comprising the upstream promoter is flanked by a 200 base pair long inverted repeat with stretches of homology to other regions of the X. laevis genome, we speculate that it might have been inserted upstream of the vitellogenin gene A1 by a recombination event and consequently brought under control of the ERE lying 1.5 kilobases downstream.

Elf-1 contributes to the function of the complex interleukin (IL)-2-responsive enhancer in the mouse IL-2 receptor alpha gene.

Lymphocytes regulate their responsiveness to IL-2 through the transcriptional control of the IL-2R alpha gene, which encodes a component of the high affinity IL-2 receptor. In the mouse IL-2R alpha gene this control is exerted via two regulatable elements, a promoter proximal region, and an IL-2-responsive enhancer (IL-2rE) 1.3 kb upstream. In vitro and in vivo functional analysis of the IL-2rE in the rodent thymic lymphoma-derived, CD4- CD8- cell line PC60 demonstrated that three separate elements, sites I, II, and III, were necessary for IL-2 responsiveness; these three sites demonstrate functional cooperation. Site III contains a consensus binding motif for members of the Ets family of transcription factors. Here we demonstrate that Elf-1, an Ets-like protein, binds to site III and participates in IL-2 responsiveness. In vitro site III forms a complex with a protein constitutively present in nuclear extracts from PC60 cells as well as from normal CD4- CD8- thymocytes. We have identified this molecule as Elf-1 according to a number of criteria. The complex possesses an identical electrophoretic mobility to that formed by recombinant Elf-1 protein and is super-shifted by anti-Elf-1 antibodies. Biotinylated IL-2rE probes precipitate Elf-1 from PC60 extracts provided site III is intact and both recombinant and PC60-derived proteins bind with the same relative affinities to different mutants of site III. In addition, by introducing mutations into the core of the site III Ets-like motif and comparing the corresponding effects on the in vitro binding of Elf-1 and the in vivo IL-2rE activity, we provide strong evidence that Elf-1 is directly involved in IL-2 responsiveness. The nature of the functional cooperativity observed between Elf-1 and the factors binding sites I and II remains unresolved; experiments presented here however suggest that this effect may not require direct interactions between the proteins binding these three elements.

The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver.

BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.

Clinical and genetic findings in a large cohort of patients with congenital myopathies due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene

RYR1 mutations are the most common cause of structural congenital myopathies and may exhibit both dominant and recessive inheritance. Histopathological findings are variable and include central cores, multi-minicores, type 1 predominance/ uniformity, fibre type disproportion, increased internal nucleation and fatty and connective tissue. Until recently, diagnostic RYR1 sequencing was limited to mutational hotspots due to the large size of the gene. Since the introduction of full RYR1 sequencing in 2007 we have detected pathogenic mutations in 77 families: 39 had dominant inheritance and 38 recessive inheritance. In some cases with presumably recessive inheritance, only one heterozygous mutation inherited from an asymptomatic parent was identified. Of 28 dominant mutations, 6 were novel; 37 of the 59 recessive mutations were also novel. Dominant mutations were more frequently in recognized hotspot regions, while recessive mutations were distributed throughout the coding sequence. Dominant mutations were predominantly missense, whereas recessive mutations included many nonsense and splice mutations expected to result in reduced RyR1 protein. There was wide clinical variability in patients with both dominant and recessive inheritance. As a group, those with dominant mutations were generally more mildly affected than those with recessive inheritance, who had earlier onset and were weaker with more functional limitations. Extraocular muscle involvement was almost exclusively observed in the recessive group. Bulbar involvement was also more prominent in this group, resulting in a larger number requiring gastrostomy insertion. In conclusion, genomic sequencing of the entire RYR1 leads to the detection of many novel mutations, but may miss large genetic rearrangements in some cases. Assigning pathogenicity to novel mutations is often difficult and interpretation of genetic results in the context of clinical, histological and, increasingly, muscle MRI findings is essential.