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Thesis Reference Thesis Exploring regulation and expression of the Abd-B Homeotic Gene using BAC technology in Drosophila: a new role in reproduction GLIGOROV, Dragan Abstract We discovered that the hox gene Abdominal B is specifically expressed in the secondary cells of the Drosophila male accessory gland. Using an Abd-B BAC reporter coupled with a collection of genetic deletions, we discovered an enhancer in the iab-6 regulatory domain that is responsible for Abd-B expression in these cells removal of which results in visible morphological defects in the secondary cells. Mates of iab-6 mutant males show defects in long-term egg laying and suppression of receptivity phenotypes. These data for the first time uncovered part of the function of the secondary cells. Transcriptome analysis from WT and mutant accessory glands led us to consider a list of 73 target genes that are down regulated in the mutant gland. The results RNAi experiments on these genes are presented in detail within the thesis. Interesting case of transvection as well as Gal4 toxicity are also included in these thesis. Reference GLIGOROV, Dragan. Exploring regulation and expression of the Abd-B Homeotic Gene using BAC technology in Drosophila: a new role in reproduction. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4562 URN : urn:nbn:ch:unige-290620 DOI : 10.13097/archive-ouverte/unige:29062 Available at: http://archive-ouverte.unige.ch/unige:29062 Disclaimer: layout of this document may differ from the published version. 1 / 1 UNIVERSITE de GENEVE FACULTE DES SCIENCES Département de génétique et evolution Professeur François Karch Exploring Regulation and Expression of the Abd-B Homeotic Gene Using BAC Technology in Drosophila: A New Role in Reproduction THESE Présentée à la Faculté des Sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie par Dragan Gligorov de Macédoine Thèse No – 4562 - Genève Atelier d’impression ReproMail Mai 2013 Table of content Résumé français 3 Summary 7 Introduction 11 Abd-B regulation 11 The domain model 14 Long-distance interaction in the BX-C 18 Transvection 18 Boundary Elements in Long-Distance Chromatin Interactions 20 Results 24 Part I. Ectopic trans-activation of Abd-B in the salivary gland by the 24 Abd-B Gal4 Bac Adult salivary gland shows ectopic Abd-B expression 26 DNA FISH 28 Salivary gland morphological phenotype 30 Secondary cell enhancer studies suggest Gal-4 toxicity 35 Conclusion 41 Part II. A novel function for Abd-B in the male accessory gland 43 Creation of BAC reporters/fusions for Abdominal-B 47 Previously unknown location of Abd-B expression in adult flies 51 Secondary cell enhancer 55 Abd-B expression in secondary cells is independent of the initiator 61 Part III. Dissection of the secondary cell transcriptome 62 RNAi 64 Preliminary in situ hybridization results 69 Abd-Bm ISH 70 Antibody development 71 1 Vacuolar markers 71 Discussion 74 New insights into Abd-B regulation 74 Initial observation from the mRNA-seq data 77 Possible crosstalk between the different cell types in the 79 male reproductive system Cluster or group of genes are simultaneously regulated 80 RNAi of candidate genes 81 The vacuoles 83 Abd-Bm in situ hybridization 84 My Summary 86 Materials and Methods 89 Appendix 113 Table 1. The 73 candidate genes, predicted molecular function, maximal tissue 113 expression Table 2. RNAi experiment egg laying and secondary cell morphological data 116 References 120 My First Paper ( added as a pdf print out from the journal PLOS Genetic with supplementary figures ) After page. A novel function for the hox gene abd-B in the male accessory gland regulates the long-term 130 female post-mating response in Drosophila. 2 Résumé français Les gènes homéotiques (également connus sous le nom de gènes architectes, ou gènes Hox) sont connus pour le rôle qu’ils jouent dans la détermination des structures qui se forment le long de l’axe antéropostérieur des organismes à symétrie bilatérale. C’est l’analyse génétique chez la drosophile ( Drosophila melanogaster) qui a conduit à l’identification des gènes homéotiques, grâce à la découverte de mutations qui transforment les antennes des drosophiles en pattes, ou des mutations qui conduisent à l’apparition de mouches avec 2 paires d’ailes au lieu d’une seule. De façon très intrigante, les gènes architectes sont arrangés le long du chromosome dans le même ordre que les segments qu’ils spécifient le long de l’axe antéropostérieur. De façon encore plus remarquable cette correspondance entre organisation génomique et axe antéro-postérieur a été conservée au cours de l’évolution, et des complexes similaires de gènes architectes (ou complexes Hox) se retrouvent chez les vertébrés, les mammifères et chez l’homme. Au cours de mon travail de thèse j’ai étudié la régulation du gène homéotique Abdominal-B (Abd-B) qui appartient au complexe bithorax et qui détermine l’identité des segments abdominaux postérieur de la drosophile (A5 à A8). Le gène Abd-B est très complexe et occupe avec ses séquences régulatrices beaucoup d’espace sur le chromosome. Afin de mieux comprendre son mode de régulation, nous avons créer un chromosome artificiel d’origine bactérienne qui exprime une protéine fluorescente de méduse (GFP) dans le contexte génomique normal d’Abd-B. L’expression de la GFP mimant celle du gène Abd-B, peut alors être suivie in vivo, en observant des embryons, larves ou mouches adultes à la lumière ultra-violette. J’ai ainsi découvert, que le 3 gène Abd-B est exprimé dans les « glandes accessoires » de l’appareil reproductif mâle. Ces « glandes accessoires », (qui pourraient s’apparenter à la prostate chez l’homme), synthétisent un cocktail de peptides qui composent le liquide séminal et qui modifient le comportement des femelles une fois l’accouplement terminé. Lors de l’accouplement, la drosophile femelle reçoit le sperme qu’elle stocke dans des structures spécialisées, les spermathèques et les glandes séminales, pour féconder de manière autonome les nombreux ovoyctes qu’elle libère durant la dizaine de jours suivants. Ce sperme contient en plus des spermatozoïdes, des composés, parmi lesquels le « sex peptide » qui induit chez la femelle, des transformations physiologiques telles que la stimulation de l’ovogenèse ou du système immunitaire, ainsi qu’un changement de comportement. Les femelles fraichement fécondées repoussent les sollicitations d’autres mâles. En d’autres termes, en transférant son « sex petide », le male s’assure de l’exclusivité de la transmission de ses propres gènes. Les « glandes accessoires » sont des organes tubulaires composés d’un millier de cellules d’origine mésodermale (les cellules principales). A l’extrémité distale de la glande se trouvent une soixantaine de cellules (dites secondaires) à la morphologie clairement distincte du reste des cellules de la glande, par la présence de grandes vacuoles dans leur cytoplasme. Le gène Abd-B est exprimé spécifiquement dans ces cellules secondaires. En puisant dans la collection d’allèles du laboratoire , j’ai identifié une mutation qui élimine l’expression d’ Abd-B dans les cellules secondaires des glandes accessoires. La perte de l’expression d’ Abd-B dans ces cellules ne cause pas leur transformation en cellules principales. Le gène Abd-B n’agit donc pas comme déterminant de l’identité cellulaire dans les glandes accessoires. Les cellules secondaires restent secondaires, mais les vacuoles présentes dans leur cytoplasme disparaissent. Cette mutation n’a aucune autre conséquence sur la spécification des segments abdominaux et les mouches éclosent avec une morphologie externe 4 parfaitement normale.. En collaboration avec le laboratoire de Mariana Wolfner de l’Université de Cornell dans l’état de New York, nous avons démontré que les femelles fécondées par les males mutants ne repoussent plus les nouveaux courtisans après un accouplement. En référence à ce phénotype cet allèle a été baptisé iab-6cocu . Le « sex peptide » qui est synthétisé par les cellules principales est bien transmis aux femelles. Mais, s’il reste détectable pendant une dizaine de jours chez les femelles fécondées par des males normaux, le « sex peptide » disparaît beaucoup plus rapidement des femelles fécondées par des males iab-6cocu . Cette découverte a une certaine résonnance dans le petit monde intéressé par la question du changement de comportement des femelles suite à l’accouplement, car elle démontre l’importance des cellules secondaires des glandes accessoires. L’existence de deux types cellulaires distincts dans les glandes accessoires, cellules primaires et secondaires et l’expression du gène Abd-B uniquement dans ces dernières, suggère un mécanisme de compartimentation. On peut imaginer que le liquide séminal doit être activé lors de l’éjaculation, pour que le « sex petide » soit stabilisé une fois transféré à la femelle.. Des expériences dites de « proteomic » ont permis l’identification d’environ 180 protéines spécifiques des glandes annexes (appelées Acps pour « Accessory gland-specific proteins). Avec notre nouvelle lignée exprimant la GFP spécifiquement dans les cellules secondaires et l’identification de la mutation qui élimine l’expression d’ Abd-B dans ces mêmes cellules, le laboratoire dispose de nouveaux outils pour identifier les Acps spécifiques des cellules secondaires et ainsi mieux comprendre la complexité de la biologie de la reproduction chez la drosophile. Ces observations pourraient également apporter un éclairage sur des questions évolutives. L’arrangement des gènes homéotiques au sein des complexes Hox reflète l’ordre des segments ou métamères dans lesquels ils sont actifs. Le gène Abd-B et ses homologues chez les vertébrés 5 (hox10-13) sont positionnés à l’extrémité des complexes Hox active dans les segments ou métamères postérieurs de l’embryon. Chez la drosophile, le gène Abd-B est actif dans les segments abdominaux 5 à 8 et dans la « primordia » qui va donner naissance aux structures génitales externes.
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