Pineal expression-promoting element (PIPE), a cis- acting element, directs pineal-specific gene expression in zebrafish

Abstract

The pineal gland, sharing morphological and biochemical similarities with the retina, plays a unique and central role in the photoneuroendocrine system. The unique development of the pineal gland is directed by a specific combination of the expressed genes, but little is known about the regulatory mechanism underlying the pineal-specific gene expression. We isolated a 1.1-kbp fragment upstream of the zebrafish exo-rhodopsin (exorh) gene, which is expressed specifically in the pineal gland. Transgenic analysis using an enhanced green fluorescent protein reporter gene demonstrated that the proximal 147-bp region of the exorh promoter is sufficient to direct pineal-specific expression. This region contains three copies of a putative cone rod homeobox (Crx)͞Otx-binding site, which is known to be required for expression of both retina-and pineal-specific genes. Deletion and mutational analyses of the exorh promoter revealed that a previously uncharacterized sequence TGACCCCAATCT termed pineal expression-promoting element (PIPE) is required for pineal-specific promoter activity in addition to the Crx͞Otx-binding sites. By using the zebrafish rhodopsin (rh) promoter that drives retina-specific expression, we created a reporter construct having ectopic PIPE in the rh promoter at a position equivalent to that in the exorh promoter by introducing five nucleotide changes. Such a slight modification in the rh promoter induced ectopic enhanced green fluorescent protein expression in the pineal gland without affecting its retinal expression. These results identify PIPE as a critical cis-element contributing to the pineal-specific gene expression, in combination with the Crx͞Otx-binding site(s).

Novel functions for Period 3 and Exo-rhodopsin in rhythmic transcription and melatonin biosynthesis within the zebrafish pineal organ

“…The pineal opsin protein Exorh is expressed in pineal photoreceptors from early embryogenesis to adulthood and is thus an excellent candidate to be mediating these light responses [5,38,60]. Consistent with this, the action spectrum of acute melatonin suppression in isolated adult pineal organs suggests that Exorh is one of several opsins involved in this process [65].…”

“…The exorh gene is expressed in pineal photoreceptors from approximately 18 hours post fertilization (hpf) to adulthood [5,19,38,60]. Vuilleumier and colleagues recently reported that exorh is expressed with a daily rhythm, with higher expression during the dark phase of the circadian cycle [60].…”

“…The exorh promoter contains three putative Otx binding sites 5' to the translation start site. Mutation of these sites attenuates the ability of the exorh promoter to drive transgene expression in the pineal [5].…”

“…To determine whether Otx5 is capable of driving exorh expression in cells outside of the pineal, we took advantage of the exorh:Green Fluorescent Protein (exorh:GFP) transgenic line [5]. Single blastomeres of 8-16 cell stage zebrafish embryos were co-injected with otx5 and Red Fluorescent Protein (RFP) mRNA.…”

“…Since Exorh protein is likely involved in pineal photoreception, this raises the possibility that Exorh could regulate the expression of its own gene. To test this, we injected exorh sp MO into embryos carrying the exorh:GFP transgene [5]. As this transgene does not contain the exorh sp MO binding site, its expression cannot be directly affected by the injections.…”