Università degli Studi di Ferrara DOTTORATO DI RICERCA IN BIOLOGIA EVOLUZIONISTICA E AMBIENTALE CICLO XXVII COORDINATORE Prof. GUIDO BARBUJANI Molecular and Bioinformatic Analysis of the Circadian Clock in Phreatichthys andruzzii Settore Scientifico Disciplinare BIO/05 Dottorando Tutore Dott. NEGRINI PIETRO Prof. BERTOLUCCI CRISTIANO Anni 2012/2014 2 TABLE OF CONTENTS Page Abstract (English)...…………………………………………………............................7 Abstract (Italiano)...........................................................................................................9 I. Introduction………………………………………………....................................11 1.1 General aspects of the circadian clock……………………………………….13 1.1.1 Circadian pacemakers………………………..........................................................13 1.1.2 Molecular clock mechanism…………………………………………………….…15 1.1.3 Light input pathways……………………………………………………...…….…19 1.1.4 Temperature input pathway………………………………………...……………..21 1.1.5 Output pathways…………………………………………………………………...22 1.2 Zebrafish as a circadian clock model system……………...………………22 1.2.1 Zebrafish circadian clock genes…………………………………………….……..24 1.2.2 Zebrafish light input pathway……………………………………………………..24 1.2.3 Zebrafish temperature input pathway…………..…………………………..……27 1.2.4 The role of the SCN and pineal gland in zebrafish…….…………………..…….27 1.2.5 Circadian clock outputs in zebrafish………………………………………….…..28 1.3 Cavefish as a new circadian clock model system………………..…….….29 1.3.1 Life in the darkness: special features of cave inhabitants………………….…....29 1.3.2 Astyanax mexicanus: a teleost cavefish model…………………………………....30 1.3.3 The Somalian cavefish Phreatichthys andruzzii…………………………………..33 1.4 Aim of the study…………………………………………………………………..35 II. Materials and methods...................................................................................37 2.1 Animals……………..................................................................................................37 2.2 Cell lines.....................................................................................................................37 2.3 Genomic DNA extraction....................................................................................38 2.4 RNA extraction and reverse transcription....................................................38 3 2.5 Cloning Period2 sequence....................................................................................39 2.6 RT-PCR reactions………………………………………………………………..39 2.7 Quantitative PCR (qPCR)………………………………………………….......40 2.8 Recording of adult locomotor activity………………………………………41 2.9 Deep Sequencing analysis…………………………………………………..…..42 2.9.1 Stranded RNA sequencing libraries construction………………………....……..42 2.9.2 Paired-end DNA sequencing construction………………………………………..42 2.9.3 Mate pair libraries construction……………………………..………….….……..43 2.9.4 Sequencing of libraries……………………………………………………....……..43 2.9.5 De novo transcriptome assembly…………………………………………….…….44 2.9.6 De novo genome assembly………………………………………………….……....44 III. Results....................................................................................................................46 3.1 Cavefish Period2 analysis……………………………………………………....46 3.1.1 Cloning cavefish Period2 gene…………………………………………...….……..46 3.1.2 Transposon validation and expression levels of cavefish Per2/Cry1a…………...50 3.1.3 Next Generation Sequencing: a new global approach………………………..….52 3.2 RNAseq analysis in Phreatichthys andruzzii……..…...…………………....52 3.2.1 General statistics on Phreatichthys andruzzii RNAseq……….……….………….52 3.2.2 The characterization of cavefish circadian oscillator from RNAseq…..………..54 3.3 Intron retention in cavefish mRNA………….……………..………..………56 3.4 DNAseq analysis in Phreatichthys andruzzii……….……………………....62 3.4.1 General statistics on Phreatichthys andruzzii DNAseq……….………....………..62 3.4.2 The analysis of the visual/non-visual opsins from the cavefish genome……...…63 4 IV. Discussion.............................................................................................................68 4.1 Cavefish Period2: a candidate underlying the aberrant circadian clock properties………….………………………...…...68 4.2 RNAseq analysis and intron retention in Phreatichthys andruzzii mRNA…………………………………...…………….69 4.3 Genomic sequencing and visual/non-visual opsins analysis in Phreatichthys andruzzii…………………….…………………………..…….…...72 4.4 Phreatichthys andruzzii and its aberrant clock phenotype………...…...74 V. References...............................................................................................................77 Acknowledgements………………………………………………………………102 5 6 Abstract (english) Daily cycles of light and temperature imposed by the rotation of the Earth on its axis have had a major impact on the evolution of all living organisms. Fascinating demonstrations of this fact can be seen in extreme environments such as caves or in the deep sea, where some species have evolved in complete isolation from daily light-dark cycles for millions of years, sharing a range of striking physical characters acquired by convergent evolution such as loss of eyes and pigmentation. One fundamental issue is to investigate whether these “hypogean” species still retain a functional circadian clock, which is a highly conserved self-sustaining timing system that allows organisms to anticipate daily environmental changes and is synchronized primarily by light. In this study, we have performed a comparative analysis of the circadian clock between the Somalian cavefish Phreatichthys andruzzii, which has evolved in perpetual darkness, and the model species Danio rerio (the zebrafish) that is evolved under natural daily light-dark cycles. It has been demonstrated that P. andruzzii retains a food-entrainable clock that is synchronized in response to regular feeding time, but does not respond to light-dark cycles. Moreover, under constant conditions, the cavefish clock oscillates with an extremely long period and also lacks normal temperature compensation. Based on these previous results, we started to analyze in detail one specific clock gene that is light-induced in zebrafish, Period2, where we encountered significant mutations in cavefish. We characterized the coding sequence and the genomic structure of this mutated cavefish gene and we analyzed its expression levels in comparison with zebrafish. Subsequently, for the first time in this species we performed a detailed characterization of clock and visual/non-visual photoreceptor genes as part of a complete P. andruzzii genome and transcriptome analysis. Our RNAseq analysis revealed a surprising phenomenon in cavefish: P. andruzzii mRNA sequences present an unusually high level of retained introns, leading to premature stop codons being introduced into the coding sequences of many transcripts. This mechanism may contribute to the aberrant clock phenotype of this species as well as, from a wider perspective, to other fascinating cavefish adaptations to life in constant darkness. We analyzed in detail this aberrant intron splicing phenomenon in two cavefish transcriptomes, from the brain and from a fin-derived cell line. Finally, the creation of a first P. andruzzii genome assembly by DNAseq analysis allowed us to map and characterize the group of visual/non- visual opsins, the main candidate genes involved in the circadian and non- photoreception 7 system. We used this sequence data in order to perform a comparative analysis of the levels of expression of these genes between cavefish and zebrafish. Our results suggest that the loss of photoreceptor function in cavefish may result either from mutations affecting the coding regions of the opsins, as documented in TMT-opsin and Opn4m2, or, alternatively, from mutations affecting the regulation of the expression levels of this group of photoreceptive genes. Keywords: circadian clock, cavefish, zebrafish, Period2 gene, transcriptome, genome, de novo assembly, intron splicing, visual and non-visual ospins. 8 Abstract (Italiano) I cicli giornalieri di luce e temperatura imposti dalla rotazione della Terra sul suo asse hanno avuto un forte impatto sull'evoluzione di tutti gli organismi viventi. Dimostrazioni affascinanti di questo fatto si possono trovare in ambienti estremi come grotte o abissi marini, in cui alcune specie si sono evolute durante milioni di anni in completo isolamento dai cicli giornalieri di luce e buio, acquisendo un’ampia gamma di caratteri fisici come la perdita degli occhi e della pigmentazione attraverso un’evoluzione convergente. Un problema fondamentale è quello di verificare se queste specie ipogee posseggano ancora un orologio circadiano funzionale, cioè un sistema endogeno altamente conservato che permette agli organismi di anticipare i cambiamenti ambientali quotidiani e che è sincronizzato principalmente dalla luce. In questo studio, abbiamo eseguito un'analisi comparativa del sistema circadiano tra il pesce ipogeo della Somalia Phreatichthys andruzzii, evolutosi in buio costante, e la specie modello Danio rerio (lo zebrafish) la quale è normalmente esposta ai cicli giornalieri di luce e buio. È stato dimostrato che P. andruzzii possiede un orologio circadiano sincronizzabile in risposta a somministrazione regolare di cibo, ma non a cicli di luce-buio. Inoltre, in condizioni costanti, l'orologio di questo pesce ipogeo si esprime con un periodo estremamente lungo e manca della normale compensazione del periodo al variare della temperatura. Partendo da questi risultati precedentemente ottenuti, abbiamo analizzato in dettaglio