In vivo analysis of zebrafish exo-rhodopsin protein and suprachiasmatic nucleus function

The zebrafish pineal is a powerful system to dissect the regulation of circadian rhythms during development. It has all the elements of the circadian timing system. Under constant conditions, an endogenous pineal clock drives many rhythms including higher levels of circulating melatonin at night and lower levels during the day. Photoreceptors within the zebrafish pineal reset the endogenous circadian oscillator. It has been shown that light is partly required for higher levels of mRNA expression of two rhythmic pineal genes extra-ocular rhodopsin (exorh) and serotonin-N-acetyltransferase 2 (aanat2) that encodes for a putative photopigment and a major melatonin biosynthetic enzyme, respectively.;Many candidate photopigments are expressed in the zebrafish pineal such as red opsin, blue opsin, and Exorh. To determine the role of Exorh protein, I used two non-overlapping antisense morpholino oligonucleotides (MOs) to deplete Exorh. I found a dramatic reduction in the levels of exorh mRNA in Exorh-deficient embryos. Together with my fellow graduate student Lain Pierce, I discovered significantly reduced levels of aanat2 mRNA in Exorh-deficient embryos. Since higher levels of exorh and aanat2 mRNAs are partly dependent on light, these results suggest that Exorh participates in a light-sensing pathway that ultimately causes the activation of pineal gene expression. This is the first evidence for Exorh function.;In mammals, pineal rhythms are regulated by the suprachiasmatic nucleus (SCN) of the hypothalamus. While a similar structure has been identified in teleost fish such as zebrafish, evidence for its function is lacking. To define the role of the zebrafish SCN, I analyzed pineal gene expression rhythms in cyclops (cyc) mutants. cyc mutants are missing the ventral brain including the SCN precursors. My fellow graduate student Po-Nien Lu and I showed that the rhythmic expression patterns of aanat2 and exorh are indistinguishable between wild type (WT) fish and cyc mutants. Specifically, the timing of high and low levels of gene expression is similar in WT and cyc mutants. Together, these results suggest that the zebrafish SCN has at most a minor role in generating pineal rhythms. This is the first in vivo test for zebrafish SCN function.