ANALYSIS OF RHYTHMIC GENE TRANSCRIPTION USING THE TimeR, A NOVEL TECHNOLOGY TO CAPTURE ZEBRAFISH EMBRYOS by LAIN XYLIA PIERCE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Jennifer O. Liang, PhD Department of Genetics CASE WESTERN RESERVE UNIVERSITY August 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. 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Table of Contents Table of Contents 1 List of Tables 3 List of Figures 3 Abstract 5 Chapter 1: Background and Significance Circadian Rhythms 7 The Importance of Circadian Rhythms in Humans 8 The Clock, Cell Division and Cancer 10 The Vertebrate Circadian System 12 The Mammalian Molecular Clock 13 The Zebrafish Molecular Clock 15 Photoreception and Circadian Entrainment 16 Studying the Circadian Clock 17 Summary of Scientific Contributions 18 Chapter 2: The Time Reaper 5­channel Automatic Liquid Dispenser: a new tool for studying zebrafish development Abstract 21 Introduction 22 Results and Discussion 23 Design and use of the TimeR 23 1 The TimeR is an effective tool for fixing zebrafish embryos 25 Materials and Methods 27 Acknowledgements 29 Chapter3: Novel functions for Period 3 and Exo­rhodopsin in rhythmic transcription and melatonin biosynthesis within the zebrafish pineal organ Abstract 43 Introduction 44 Results 45 exorh transcription is rhythmic 45 Otx5 activates exorh transcription 46 Per3 negatively regulates exorh transcription during the day 47 Loss of Exorh protein reduces transcription from the exorh promoter 48 Exorh does not initiate transcription of red opsin 50 Exorh is required for high levels of aanat2 expression 50 Discussion 50 Exorh protein is important for gene transcription in the zebrafish pineal organ 50 The pattern of exorh transcription is controlled by a combination of tissue­specific and rhythmic factors 51 Experimental Procedures 55 Acknowledgements 57 2 Chapter 4: Discussion and Future Directions Novel function for Exorh in regulating gene transcription in the zebrafish pineal organ 86 Novel function for Per3 in regulating the phase of expression for a rhythmic gene. Per3 had been identified but function was not well understood 88 Rhythmic genes are regulated by tissue specific factors that restrict special expression and rhythmic factors that generate the daily changes in mRNA levels 91 Connections between phototransduction and circadian rhythms 94 References 100 3 List of Tables Chapter 2 Supplementary Parts List 51 Chapter 3 Table 1: Overexpression of Otx5 induces ectopic expression of the exorh:GFP transgene 70 Supplementary Table 1: Analysis of exorh expression using the Student’s t‐test 85 4 List of Figures Chapter 1 Figure 1: The Vertebrate Circadian System 24 Figure 2: The Mammalian Circadian Clock 26 Figure 3: Alignment of amino acid sequences across visual and pineal Opsins 28 Figure 4: Phylogenetic tree comparing zebrafish exorhodopsin to other vertebrate Opsins 30 Chapter 2 Figure 1: The TimeR 42 Figure 2: Flow of liquids through the TimeR 44 Figure 3: The strength and pattern of the signal from WISH is indistinguishable between embryos fixed with the TimeR and embryos fixed manually 46 Figure 4: Whole mount immunostaining on TimeR and manually fixed embryos 48 Figure 5: Temperature of fixative does not diminish the effectiveness or accuracy of either WISH or Immunostaining 50 Chapter 3 Figure 1. There are significant changes in exorh expression levels between day and night 72 Figure 2. Otx5 controls the tissue‐spec ificity of exorh transcription 74 Figure 3. Daytime expression of exorh is increased in embryos lacking Per3 76 5 Chapter 3 Figures cont. Figure 4. Expression from the exorh promoter is decreased in embryos lacking Exorh protein 79 Figure 5. Exorh is required for high levels of aanat2 tran s cription 81 Figure 6. Model for the regulation of exorh expression 83 Supplem entary Figure 1. Exorh protein is required for exorh transcription throughout the circadian cycle 84 Chapter 4 Figure 1. The Molecular Pathway Between Light and Transcription 97 Figure 2: Conclusions and Future Directions 99 6 Analysis of Rhythmic Gene Transcription using the TimeR, a Novel Technology to Capture Zebrafish Embryos Abstract by LAIN XYLIA PIERCE Circadian rhythms are predictable oscillations in behavior such as the sleep wake cycle and biomolecular profiles such as rhythmic changes in gene transcription. These oscillations receive entraining cues from environmental inputs such as the light/dark photoperiod that regulate endogenous molecular clocks which in turn produce rhythmic outputs. The zebrafish pineal organ is an excellent model to study these molecular profiles because phototransducive molecules as well as the clock and its outputs are all housed within the same cells. Additionally, zebrafish embryos undergo rapid and external development such that the molecular pineal clock is functional in as little as 24 hours post fertilization. However, capturing embryonic samples at all time points in the circadian cycle can be challenging. Not only is the health of the researcher compromised when working odd hours but experimental results may be altered by handling samples at inappropriate times. To address this issue, I have led the research and design of a new technology. 7 The Time Reaper 5‐Channel Automatic Liquid Dispenser (TimeR) delivers up to 50 mL of paraformaldehyde to embryos in a Petri dish at preset times. In testing the TimeR I have shown that the pattern of expression for a number of genes is indistinguishable between embryos fixed manually versus remotely and that the TimeR is also suitable for fixing embryos for whole mount immunostaining. The TimeR was used frequently in securing data for the body of my dissertation work. The zebrafish opsin protein Exo‐rhodopsin (Exorh) is expressed in pineal photoreceptors and is a candidate to mediate the effects of environmental light on pineal rhythms and melatonin synthesis. We demonstrate that Exorh has an important role in regulating gene transcription within the pineal. In developing embryos that lack Exorh, expression of the exorh gene itself and of the melatonin synthesis gene serotonin N‐acetyl transferase 2 (aanat2) are significantly reduced. This suggests that Exorh protein at the cell membrane is part of a signaling pathway that positively regulates transcription of these genes, and ultimately melatonin production, in the pineal. Like many other opsin genes, exorh is expressed with a daily rhythm: mRNA levels are higher at night than during the day. We find that the transcription factor Orthodenticle homeobox 5 (Otx5) activates exorh transcription, while the putative circadian clock component Period 3 (Per3) represses expression during the day, thereby contributing to the rhythm of transcription. This work identifies novel roles for Exorh and Per3, and gives insight into potential interactions between the sensory and circadian systems within the pineal. 8 Chapter 1: Background and Significance Circadian Rhythms Circadian rhythms are biochemical and behavioral changes that occur with a period of approximately 24 hours. For example, these biorhythms control the timing of sleeping, waking, feeding and socializing. The oscillations of the clocks that drive these rhythms are self‐driven and can be steadily maintained without environmental influence. Thus, circadian rhythms allow animals to not only respond to the environment but to anticipate environmental changes. Daido(Daido, 2002) and Michael and colleagues (Michael and McClung, 2003; Michael et al., 2003) theorize that clock entrainment produces evolutionary fitness, which may explain the fact that nearly every organism studied to date possesses a circadian clock. The circadian clock is a self‐sustained oscillator driven by a set of feed‐back loops. One full revolution of the clock requires approximately 24 hours to be completed. However, the word “approximately” cannot be overstated. The endogenous human clock takes 24 hours 11 minutes ±16 minutes to complete a revolution(Czeisler et al., 1999). Clearly, if left unadjusted the molecular clock would slowly drift in and out of synchronization with the surrounding environment. Therefore, the timing of the clock contains a certain amount of plasticity. The clock can re‐set its position in the cycle in response to daily environmental cues, with the most powerful cue being photoperiod. This daily coordination is called “entrainment” and allows the molecular and physical behaviors of an organism to mirror appropriate environmental conditions. Clock plasticity is also beneficial 9 when considering adaptation to changes in day length due to the changing seasons or migration and how day length affects physical security, food and mate availability. Thus, some of the same mechanisms, such as the hormone melatonin, are involved in both circadian and seasonal rhythms (Wehr, 1997). The Importance of Circadian Rhythms in Humans Over the past few years the