Characterization of the Molecular Clockwork in the Cockroach Rhyparobia Maderae
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Characterization of the molecular clockwork in the cockroach Rhyparobia maderae The core feedback loop genes period, timeless 1 and cryptochrome 2 Dissertation zur Erlangung des akademischen Grades Doktor der Naturwissenschaften (Dr. rer. nat.) Vorgelegt im Fachbereich Mathematik und Naturwissenschaften der Universit¨at Kassel von Achim Werckenthin Kassel, Dezember 2013 Eidesstattliche Erkla¨rung Hiermit versichere ich, dass ich die vorliegende Dissertation selbstst¨andig, ohne unerlaubte Hilfe Dritter angefertigt und andere als die in der Dissertation angegebenen Hilfsmittel nicht benutzt habe. Alle Stellen, die w¨ortlich oder sinngem¨aß aus ver¨offentlichten oder unver¨offentlichten Schriften entnommen sind, habe ich als solche kenntlich gemacht. Dritte waren an der inhaltlich- materiellen Erstellung der Dissertation nicht beteiligt; insbesondere habe ich hierf¨ur nicht die Hilfe eines Promotionsberaters in Anspruch genommen. Kein Teil dieser Arbeit ist in einem anderen Promotions- oder Habilitationsverfahren verwendet worden. Kassel, 13.12.2013 Achim Werckenthin iii iv Vom Fachbereich 10, Mathematik und Naturwissenschaften der Universitat¨ Kassel als Dissertation am 13.12.2013 angenommen. Prufungskommission¨ 1. Gutachterin: Prof. Dr. Monika Stengl 2. Gutachterin: Prof. Dr. Mireille Schafer¨ 3. Gutachterin: Prof. Dr. Charlotte Forster¨ 4. Gutacher: Prof. Dr. Friedrich Herberg Tag der mundlichen¨ Prufung:¨ 17.02.2014 Contents Contribution statements ix Zusammenfassung xi 1 Summary 1 2 Introduction 3 2.1 Rhyparobia maderae andcircadianresearch . 5 2.1.1 Thecentralnervoussystem. 6 2.2 Molecularrhythmgeneration . 11 2.2.1 Transcriptional-translational feedback regulation and adjacent pathways in D. melanogaster ............................. 12 2.2.2 The circadian molecular clockworkinnon-Drosophilidinsects . 16 2.2.3 Period.................................... 20 2.2.4 Timeless .................................. 22 2.2.5 Cryptochrome ............................... 23 2.2.6 Posttranslationalregulation. 26 2.3 Thecellularbasisforcircadianrhythmicity . 27 2.3.1 Clock neurons in Drosophila melanogaster ................ 27 2.3.2 The circadian pacemaker controlling locomotor rhythms in R. maderae . 29 2.3.3 Circadian oscillators outside the clock neurons and in peripheral tissues . 31 2.4 Photoperiodism ................................... 33 2.4.1 Photoperiodandcircadianrhythm . 34 2.4.2 Circadianclockgenesandphotoperiodism . 36 2.4.3 Photoperiodismincockroaches . 37 2.5 Aimsofthisstudy.................................. 39 3 Materials and methods 41 3.1 Animalrearing ................................... 41 3.2 Molecularcloning.................................. 41 3.2.1 RNAextraction............................... 42 3.2.2 cDNAsynthesisforPCRusingdegenerateprimerpairs . 42 3.2.3 Poly-A+ purification ............................ 43 3.2.4 cDNAsynthesis............................... 44 3.2.5 Adaptorligation .............................. 47 3.2.6 Primers ................................... 48 3.2.7 InitialPCR ................................. 49 3.2.8 RACE-PCR................................. 50 3.2.9 Electrophoresis ............................... 51 v vi CONTENTS 3.2.10 Gelextraction................................ 51 3.2.11 Cloning................................... 54 3.2.12 Sequencingandsequenceanalysis . 58 3.2.13 Motifprediction .............................. 58 3.2.14 Phylogeneticanalysis. 58 3.2.15 Solutions .................................. 59 3.3 Quantitativerealtimepolymerasechainreaction. 62 3.3.1 Tissuesampling............................... 62 3.3.2 RNAextraction............................... 62 3.3.3 cDNAsynthesis............................... 64 3.3.4 qPCR.................................... 65 3.3.5 Dataanalysis ................................ 67 3.4 Antibodygeneration ................................ 70 3.5 Westernblotting................................... 72 3.5.1 Tissuesamplingandhomogenization . 72 3.5.2 ProteinassayPierce660nm . 72 3.5.3 Polyacrylamidegelelectrophoresis . 73 3.5.4 Blotting................................... 76 3.5.5 Detection .................................. 78 3.5.6 Preadsorptionoftherabbitanti-rmPERantibody . 79 3.5.7 Stripping .................................. 79 3.5.8 Solutions .................................. 81 3.6 Immunohistochemistry . .. .. .. .. .. .. .. .. .. 83 3.6.1 Primaryantibodies ............................. 83 3.6.2 Secondaryantibodies. 83 3.6.3 Sections................................... 84 3.6.4 Whole-mounts ............................... 86 3.6.5 Liquidphasepreadsorption. 90 3.6.6 Imaging................................... 91 3.6.7 Imageprocessing.............................. 92 3.6.8 Quantification................................ 92 3.6.9 Solutions .................................. 93 3.7 Runningwheelassays................................ 94 4 Results 95 4.1 Molecularcloning.................................. 95 4.1.1 Period.................................... 95 4.1.2 Timeless1 ................................. 99 4.1.3 Cryptochrome2 .............................. 99 4.2 Putativecorefeedbackloopgeneexpression . 102 4.2.1 Putative core feedback loop gene expression in equinox light-dark cycles 102 4.2.2 Putative core feedback loop gene expression in constant darkness . 105 4.2.3 Putative core feedback loop gene expression in different photoperiods . 106 CONTENTS vii 4.3 Circadianoscillationsinproteinabundance . 111 4.3.1 PERIOD ..................................111 4.3.2 TIMELESS1................................112 4.3.3 CRYPTOCHROME2 ...........................113 4.4 AntirmPER-immunoreactivityinthenervoussystem . 115 4.4.1 AntirmPER-immunoreactivityinthe central nervoussystem . 115 4.4.2 AntirmPER-immunoreactivityintheventralnervecord . 120 4.5 Runningwheelactivityindifferentphotoperiods . 123 4.5.1 Lowlightactivity.. .. .. .. .. .. .. .. ..123 4.5.2 Brightlightactivity. .124 5 Discussion 127 5.1 Core feedback loop genes in R. maderae ......................127 5.1.1 Period....................................127 5.1.2 Cryptochrome ...............................128 5.1.3 Timeless1 .................................129 5.2 Corefeedbackloopgeneexpressionrhythms . 129 5.2.1 DailyexpressionrhythmsinLDcycles . 129 5.2.2 DailyexpressionrhythmsinDD . .130 5.2.3 CorefeedbackloopgeneexpressionintheAMe . 130 5.2.4 CorefeedbackloopgeneexpressioninMalpighiantubules . 131 5.2.5 The total expression level compared to actin ................131 5.2.6 Photoperioddependentplasticity. 132 5.3 Circadianrhythmsonproteinlevel . .133 5.3.1 PERIOD ..................................133 5.3.2 TIMELESS1................................135 5.4 DistributionofPER-ircellsinthecentralnervoussystem . 136 5.4.1 PER-irindifferentZeitgebertimes. 137 5.5 Locomotoractivityindifferentphotoperiods . 138 5.6 Conclusionandfutureperspectives. .140 6 Acknowledgements 169 A Appendix 171 viii CONTENTS Contribution statements The contributions of the author for each part of this thesis will be stated clearly as follows. Section 3.1: Molecular cloning • Designed the experiments. • Conducted the experiments. Generation of the adaptor-ligated RACE cDNA, initial cloning and RACE-PCR of rmPer, rmTim1 and rmCry2 was done partly in collaboration with Dr. Christian Derst at the Institute for Integrative Neuroanatomy, Charit´eBerlin. The author conducted the majority of the experiments. The initial primers that were used for cloning of rmPer were based on a rmPer sequence previously obtained by Julia Fischer and Jeff Hall. • Sequence and phylogenetic analysis. • Wrote the manuscript. • The results of this section are publishedin: WERCKENTHIN, A., DERST, C. & STENGL, M. Sequence and Expression of per, tim1, and cry2 genes in the Madeira cockroach Rhy- parobia maderae. Journal of Biological Rhythms 27(2012), 453–466. • Figures 4.1, 4.2, 4.4 and 4.5 also appeared in a similar form in the publication cited above, but were modified and re-rendered for this thesis. Section 3.2: Putative core feedback loop gene expression • Designed the experiments. • Conducted the experiments. Quantitative PCR was done in collaboration with Dr. Chris- tian Derst at the Institute for Integrative Neuroanatomy, Charit´eBerlin. The author con- ducted the majority of the experiments. Experiments for subsection 4.2.2 and constant darkness experiments of subsection 4.2.3 were conducted by the author at the University of Kassel and the Max-Planck-Institute for Terrestrial Microbiology in Marburg. • Analyzed the data. • Wrote the manuscript. • The results of this section are published in: WERCKENTHIN,A., DERST, C. & STENGL, M. Sequence and Expression of per, tim1, and cry2 genes in the Madeira cockroach Rhy- parobia maderae. Journal of Biological Rhythms 27(2012), 453–466. • Figure 4.21 also appeared in the publication cited above. Section 3.3: Circadian oscillations in protein abundance • Designed and conducted all experiments. ix x CONTRIBUTION STATEMENTS Section 3.4: Anti rmPER-immunoreactivity in the nervous system • Designed the experiments. • Conducted the experiments except the preadsorption experiments of the anti rmPER (rab- bit) antibody, which were conducted by Andreas Arendt. The majority of the immuno- staining experiments of subsection 4.4.1 were conducted by Azar Massah, under guidance of the author. • Imaging of all dissections, except dissections of the anti rmPER (rabbit) antibody pread- sorption experiments, which were scanned by Andreas Arendt. • Analyzed the data. Section 3.5: Running wheel activity in different photoperiods