Analysis of Rhythmic Gene Transcription using the TimeR, a Novel Technology to Capture Zebrafish Embryos

 
 
 
 

Analysis of Rhythmic Gene Transcription using the TimeR, a Novel Technology to Capture Zebrafish Embryos

Show full item record


Title: Analysis of Rhythmic Gene Transcription using the TimeR, a Novel Technology to Capture Zebrafish Embryos
Author: Pierce, Lain Xylia
Description: 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. 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.
Permanent Link: http://rave.ohiolink.edu/etdc/view?acc_num=case1212770242
http://hdl.handle.net/2374.OX/104194
Date: 2008

Files in this item

Files Size Format View

There are no files associated with this item.

This item appears in the following Collection(s)

Show full item record