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Brain Res. Author manuscript; available in PMC 2009 Aug 5.
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PMCID: PMC2615473

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


Entrainment of circadian clocks to environmental cues such as photoperiod ensures that daily biological rhythms stay in synchronization with the Earth’s rotation. The vertebrate pineal organ has a conserved role in circadian regulation as the primary source of the nocturnal hormone melatonin. In lower vertebrates, the pineal has an endogenous circadian clock as well as photoreceptive cells that regulate this clock. 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.

Keywords: Circadian rhythm, Exo-rhodopsin, Orthodenticle homeobox 5, Period 3, Pineal organ, Serotonin N-acetyl transferase, Transcriptional regulation

1. Introduction

Circadian rhythms are physiological and behavioral changes that occur with a period of approximately 24 hours. These oscillations are driven by an intracellular molecular clock and are self-sustaining even in the absence of environmental cues. However, the clock is entrained each day by light or other external stimuli. Since the period of circadian clocks is typically slightly longer or shorter than 24 hours, this entrainment has a critical role in ensuring that daily biological rhythms stay in synchronization with the world around.

In vertebrates, the pineal organ has a central role in the regulation of circadian rhythms as the primary source of circulating melatonin [53]. Melatonin is made during the night and acts to regulate circadian and seasonal rhythms, including daily sleep/wake cycles. In mammals, melatonin also feeds back to regulate the primary circadian clock located in the suprachiasmatic nucleus (SCN), and thus serves as a strong entraining factor [54]. Poor regulation of pineal melatonin rhythms has been related to sleep disorders, feelings of fatigue and confusion, and an increase in cancer risk [4,9,43,45].

Entrainment of mammalian pineal rhythms to environmental light is mediated by opsin proteins located in retinal photoreceptor and ganglion cells. These photoreceptive cells entrain the SCN clock, which then controls pineal rhythms through a multisynaptic pathway [30]. In contrast, the pineal of lower vertebrates contains photoreceptive cells that entrain a endogenous pineal circadian clock, likely through an opsin-mediated signaling cascade that is very similar to that found in retinal photoreceptors [30].

In zebrafish, pineal photoreceptors have been implicated in several responses to light, including entrainment of the circadian clock, triggering the onset of pineal rhythms during development, and mediating acute suppression of melatonin by a light pulse during the dark period [11,60,6365]. 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].

Many of the vertebrate opsin genes involved in circadian and visual photoreception are rhythmically expressed [68,13,17,2325,29,31,33,35,42,49,50,55,59]. We find that like these other opsin genes, exorh is expressed with a significant difference in day and night mRNA levels. Through in vivo over-expression and loss-of-function experiments we have identified three proteins that regulate exorh transcription. The pineal transcription factor Otx5 is required to activate exorh expression within the pinealocytes. The circadian regulated factor Period 3 (Per3), whose function in mammals has remained elusive, has an important role in influencing the timing of exorh expression. Finally, Exorh protein is required for expression of its own gene. These findings suggest a model in which tissue-specific factors and clock components act together to control the spatial and temporal pattern of exorh expression. In addition, we provide the first direct evidence for an in vivo function of Exorh. In addition to regulating transcription from its own gene, Exorh protein is required for high expression of aanat2, which encodes the penultimate enzyme in the melatonin synthesis pathway. Interestingly, the reduced expression of aanat2 in Exorh-depleted embryos closely matched aanat2 expression in embryos raised in constant darkness. This suggests that Exorh could be mediating the light-dependent initiation of aanat2 transcription and melatonin production in the developing pineal.

2. Results

exorh transcription is rhythmic

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]. However, they found that the day/night differences in expression were not statistically significant [60]. Consistent with this, we also found differences in the day and night levels of exorh transcripts (Figure 1). However, in contrast to this earlier study, we find that the level of exorh transcripts were often significantly lower during the day than during the night (Figure 1C and Supplementary Table 1). Since Vuilleumier et al. and this study both used whole mount RNA in situ hybridization (WISH), which is a semi-quantitative technique, this small discrepancy in our results is likely due to subtle differences in experimental conditions.

Figure 1
There are significant changes in exorh expression levels between day and night

Otx5 activates exorh transcription

The transcription factor Otx5 is expressed in the developing and mature pineal, and has been previously shown to be important for the expression of several circadian-regulated pineal genes [20]. Otx5 is also a strong candidate to regulate the transcription of exorh. 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 test whether Otx5 is required for exorh transcription, embryos were injected with an antisense morpholino (MO) that binds to the translation start site in the otx5 mRNA (otx5 MO). This MO has been previously shown to specifically and effectively knock down Otx5 protein levels [20]. Control embryos were injected with a MO containing 4 base pair mismatches (otx5 MIS) [20]. Injected embryos were raised in a light/dark (LD) cycle, fixed at regular intervals over a 24 hour period, and assayed for exorh expression. Control-injected embryos had a rhythmic pattern of exorh expression similar to that of uninjected fish (Compare Figure 1B, C with Figure 2A, B). In contrast, Otx5-depleted embryos had severely reduced or undetectable levels of exorh mRNA at all time points tested (Figure 2A, B). This finding indicates that Otx5 is required for exorh transcription in pineal photoreceptors.

Figure 2
Otx5 controls the tissue-specificity of exorh transcription

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. Control embryos were co-injected with β-galactosidase (β-gal) and RFP mRNA (Figure 2C). As co-injected mRNAs are inherited together, the RFP serves as a tracer to identify the progeny of the injected cell. Embryos were allowed to develop until approximately 24 hpf and then analyzed by fluorescence microscopy.

Embryos injected with RFP mRNA alone developed normally and the RFP-positive cells were scattered throughout the embryo. Further, ectopic GFP expression was present only very rarely in cells outside of the pineal (Figure 2C, Table 1). In embryos over-expressing Otx5, the RFP/Otx5-positive cells tended to be clustered together. Further, many of the red fluorescent cells were also green fluorescent, indicating that the exorh:GFP transgene was being expressed (Figure 2C, Table 1). As has been previously reported, otx5 mRNA injections also caused developmental defects, likely due to the role of Otx proteins in patterning the forebrain (Figure 2C)[20].

Table 1
Overexpression of Otx5 induces ectopic expression of the exorh:GFP transgene

Per3 negatively regulates exorh transcription during the day

otx5 mRNA is expressed at constitutively high levels, and Otx5 protein is required for the activation of pineal genes expressed at dawn, during daylight, and at night, suggesting that it is active at all times of day [20]. Thus, it is unlikely that Otx5 alone could control the rhythmic expression of exorh. Instead, we hypothesized that the timing of exorh transcription is regulated by a rhythmically expressed factor, such as a component of the pineal circadian clock.

The putative clock component Per3 is a potential candidate for this role because the per3 gene is expressed widely in the developing zebrafish brain with a strong daily rhythm that peaks at dawn [18]. To determine whether per3 expression is present within pinealocytes, the brains of 3 days post fertilization (dpf) fish were divided into left and right halves by a sagittal cut through the midline. At this stage, the pineal has begun to evaginate from the forebrain, and in bisected embryos appears as a distinct domain separated from the rest of the brain by a translucent area that may correspond to the developing saccus dorsalus (Figure 3A) [28,36]. otx5 expression can be easily distinguished within this domain (Figure 3A). per3 transcripts were also detected in the pineal domain at dawn, when per3 expression in the brain is at its highest point (Figure 3A)[18].In contrast, expression in the pineal was severely decreased at a night time point, when expression in the surrounding neural tissue was also low (Figure 3A)[18].

Figure 3
Daytime expression of exorh is increased in embryos lacking Per3

Per proteins typically regulate the timing of gene expression by repressing gene transcription [44]. Consistent with this, we found that exorh transcripts were up-regulated during the day in embryos injected with a per3 MO (Figure 3B, C). In contrast, exorh mRNA levels were unaffected at time points during the dark period or in embryos injected with a control MO containing 5 base pair mismatches (per3 MIS)(Figure 3B, C). This indicates that Per3 is required to suppress exorh mRNA levels during the light period of the circadian cycle.

aanat2 is a circadian-regulated gene that encodes an enzyme required for melatonin biosynthesis in the pineal [22]. aanat2 is expressed in a similar phase to exorh, with high mRNA levels at night and very low levels during the day [20,22]. Further, like exorh, aanat2 transcription in the pineal is activated by Otx5 [20]. However, in contrast to the results for exorh, there were no apparent effects on aanat2 transcription in Per3 depleted embryos (Figure 3B).

These data suggest that Per3 is present in the pineal, and could be acting directly within pinealocytes to influence exorh expression. The per3 promoter is able to drive circadian expression of luciferase in adult zebrafish pineal organs [27]. Thus, it is possible that the role of Per3 in regulating exorh rhythms could continue through adulthood.

Loss of Exorh protein reduces transcription from the exorh promoter

The early pineal-specific expression of Exorh makes it an excellent candidate to function as a major light-sensing photopigment in the developing pineal [19,64]. Towards testing the function of Exorh, we designed a MO that binds to the exon 1/intron 1 junction of the exorh genomic sequence (exorh sp MO). Embryos injected with exorh MO had severely reduced levels of exorh mRNA compared to embryos injected with a control MO (Figure 4A, B). Embryos were analyzed using a probe that binds to the 3’ end of the exorh open reading frame and recognizes both correctly and incorrectly spliced transcripts [38]. Thus, the reduced expression suggests that exorh transcripts were being degraded due to improper splicing, and that the exorh MO effectively knocks-down Exorh protein.

Figure 4
Expression from the exorh promoter is decreased in embryos lacking Exorh protein

The normal onset of rhythmic exorh transcription requires exposure of embryos to a light/dark or dark/light transition [60]. 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. Despite this, fluorescence in the pineal was significantly reduced in Exorh depleted embryos compared to embryos injected with a control MO (Figure 4C, D, G).

To verify this result, the experiment was repeated using a second, non-overlapping MO that binds to the translation start site of the exorh mRNA (exorh atg MO). Since start site MO bind to the target mRNA but do not directly affect the levels of transcripts, the effects of this MO could be assessed by measuring expression of the endogenous gene [40]. Consistent with the exorh sp MO results, injection of the exorh atg MO caused significantly reduced expression of exorh (Figure 4I, J, K, Supplementary Figure 1).

Although loss of opsin proteins can lead to photoreceptor cell death [26,32,34,47], we found no evidence for loss of photoreceptors in the Exorh depleted fish. The embryonic pineal is largely composed of photoreceptors [5,20,21,39]. Therefore, if photoreceptor cells were dying, there would be a significant change in the size of the pineal. However, the length and width of the pineal otx5 expression domain, which encompasses both photoreceptor cells and projection neurons, was unaltered in fish injected with either exorh MO (Figure 4E, F, H, L). Therefore, it is unlikely that the reduction in exorh:GFP transgene expression was due to loss of photoreceptor cells. Instead, this suggests that the decrease was caused by a down-regulation of exorh promoter activity.

Exorh does not initiate transcription of red opsin

Being a photoreceptive organ, the pineal in zebrafish expresses a number of opsins besides exorh, including the same RGB cone pigments found in the eye [46]. The fact that activation of an Exorh-dependent pathway is important for transcription of exorh in the pineal, suggests that initiation of this same pathway could be required for expression of other pineal opsins. To test this, we determined the effect of Exorh depletion on the expression of opsin 1 (cone pigments)long-wave-sensitive, 1 (opn1lw1), formerly known as red opsin [46]. opn1lw1 expression was not significantly different in exorh atg MO and control injected embryos (Figure 5A, C). In contrast, exorh transcripts were severely reduced in exorh atg MO injected embryos from the same experiment (Figure 5A, C).

Figure 5
Exorh is required for high levels of aanat2 transcription

Exorh is required for high levels of aanat2 expression

Expression of the melatonin biosynthetic gene aanat2 is high at night and very low during the day, accounting in part for the vastly higher levels of melatonin made during the dark period of the circadian cycle [22]. Previous work has demonstrated that the onset of aanat2 transcription in zebrafish requires exposure of embryos to a transition in lighting conditions from dark to light or from light to dark. We found that depletion of Exorh protein caused a significant reduction of the levels of aanat2 expression in developing embryos (Figure 5A, B). Again, the control experiment done in parallel showed the expected reduction in exorh transcripts following Exorh depletion (Figure 5A, B). This suggests that Exorh could be wholly or partially responsible for triggering aanat2 transcription in response to changes in lighting conditions.

3. Discussion

Exorh protein is important for gene transcription in the zebrafish pineal organ

Exorh protein shares a 70% sequence identity with the photoreceptive retinal opsin protein Rhodopsin, and is predicted to have seven transmembrane domains typical of G-protein coupled receptors [38]. These observations, together with the early pineal-specific expression of exorh, are consistent with Exorh functioning as a major light-sensing molecule in the developing zebrafish pineal.

Previous work demonstrates that expression of exorh and aanat2 in the pineal does not initiate normally when embryos are raised in constant darkness [60,64]. For instance, expression of aanat2 is reduced by 58–68% when embryos are moved to constant darkness shortly after fertilization [64]. Here, we provide some of the first evidence that Exorh is involved in this light response in developing embryos. In particular, MO-mediated depletion of Exorh protein caused a very similar reduction (>50%) as raising embryos in darkness. This suggests a mechanism wherein light received by Exorh protein initiates a signaling cascade that ultimately results in onset of high levels of aanat2 and exorh transcription (Figure 6).

Figure 6
Model for the regulation of exorh expression

The most likely role for Exorh is as a pineal photopigment. Consistent with the pineal photoreceptors being active at these early stages, other potential components of a pineal phototransduction cascade, such as the components of transducin heterotrimeric G-proteins, are also expressed in the developing pineal [10,37,52]. However, we do not yet know how the phototransduction cascade interfaces with the pineal transcriptional machinery. Previous work on the aanat2 regulation by Yoav Gothilf and colleagues gives some insight into how this might occur. The light-dependent onset of aanat2 transcription in the pineal requires Per2 [63,64]. Transcription of the per2 gene itself is induced by light [12,63,64]. Thus, Per2 has the potential to act as an intermediate between Exorh on the cell membrane and clock components such as Clock/BMAL on the aanat2 promoter (Figure 6).

The pattern of exorh transcription is controlled by a combination of tissue-specific and rhythmic factors

This study demonstrates that exorh expression has a significant daily rhythm in the embryonic and larval pineal photoreceptors of zebrafish. Further, we define the in vivo functions of three proteins, Exorh itself, Otx5, and Per3 in the regulation of the exorh expression pattern (Figure 6). We find that Exorh protein is required for normal transcription from its own promoter, suggesting that a phototransduction pathway containing Exorh regulates exorh transcription. Further, we find that Otx5 and Per3 have complementary roles. Our loss- and gain-of-function experiments strongly suggest that Otx5 functions as an activator to induce exorh transcription in the cells of the pineal. In contrast, Per3 influences the phase of expression by suppressing transcription during the day.

Many other opsin genes are also expressed with a strong daily rhythm [68,13,17,2325,29,31,33,35,42,49,50,55,59]. However, the function of this rhythmic expression is not well understood. One possibility is that transcription is rhythmic in order to drive rhythmic expression of the Exorh protein. For instance, the cyclic pattern of exorh closely matches the expression of several cone opsin genes, which peak at dusk and remain highly expressed through the dark period [23,29]. Interestingly, cone cells are not used for vision during this period of high expression. Korenbrot and Fernald proposed that it is metabolically advantageous to accumulate opsin protein during the dark period before rapid turnover begins at dawn [29], and this could be the case for Exorh as well.

Another possibility is that Exorh is required for a critical function during the night. There is some evidence to support this hypothesis, as Exorh has been implicated in the acute suppression of melatonin levels that occurs when adult zebrafish are exposed to a light pulse during the night [65]. However, rhythmic transcription is not always followed by cyclic expression or cyclic activity of the encoded protein. For instance, expression of the interphotoreceptor retinoid binding protein (irbp) gene in the zebrafish retina has a strong circadian rhythm, while protein levels remain essentially constant [16]. Similar to the case with cone opsin, it is thought the high expression of irbp mRNA during the day compensates for high turnover of IRBP protein.

Otx5 has now been shown to be required for the expression of four rhythmically expressed pineal genes (exorh, aanat2, irbp, and reverb-alpha), but not for the expression of three non-rhythmic pineal genes (otx5, cone rod homeobox (crx), and floating head) ([20] and this study). Together, this suggests that the role of Otx5 is to activate the expression of circadian genes within pinealocytes. Otx5 likely acts by binding to the promoters of its target genes, as the exorh and aanat2 promoters both have three putative Otx binding sites [1,5]. However, we think it unlikely that Otx5 is involved in generating the rhythm of expression. Instead, the evidence suggests that Otx5 is constitutively active. Otx5 depletion causes loss of exorh expression at all time points tested. Further, previous work demonstrates that Otx5 is required for the expression of genes expressed at night (aanat2), day (irbp) and dawn (reverb–alpha) [20].

In support of a role for Otx5 as a transcriptional activator of pineal genes, ectopic expression of Otx5 induces transcription from the exorh and aanat2 promoters in cells outside of the pineal and eye (this study and [1]). However, there is an interesting exception to the role of Otx5 as a transcriptional activator. Although Otx5 is expressed widely in the developing eye, it appears to have little or no role in regulating retinal genes. For instance, aanat2 and exorh are expressed only in a small subset of the Otx5 positive cells in the eye, indicating that Otx5 is not sufficient to activate expression of these genes in most retinal photoreceptors [20,60]. Similarly, depletion of Otx5 causes severely reduced pineal expression of irbp and the G protein γT1 subunit genes, while their retinal transcription remains unaffected [14,20]. Conversely, depletion of the related Otx family member Crx reduces transcription in the eye but not in the pineal [14,20,52]. A probable explanation is that other transcriptional regulators cooperate with Otx5 and Crx to control the tissue specificity of transcription. As evidence for this, Asaoka and colleagues have identified an element (Pineal expression-promoting element; PIPE) within the exorh promoter that confers the ability to drive pineal expression on the normally retina-specific rhodopsin promoter [5]. The protein(s) that binds to this element has not yet been identified.

Here, we also identify a novel role for Per3 in the regulation of exorh transcriptional rhythms, with loss of Per3 causing increased expression specifically during the light period of the circadian cycle. While the functions of vertebrate Per1 and Per2 proteins are well established, the function of Per3 has been difficult to define. In mice, Per1 and Per2 have central roles in the feedback loops of the circadian clock and in resetting the clock in response to environmental light cues [44]. In contrast, per3 knock out mice have only a slight decrease in the period of their circadian clock [51]. In humans, certain polymorphisms in the per3 gene and misregulation of per3 expression have been associated with breast cancer, chronic myeloid leukemia, bipolar disorder, and the structure of the sleep/wake cycle [15,41,58,61,62].

The fact that per3 mRNA is present within pinealocytes raises the possibility that Per3 protein could be acting cell autonomously to effect these changes. However, we do not yet understand the biochemical basis of Per3 activity in exorh regulation. Per proteins typically act to repress the transcriptional activity of Clock/BMAL heterodimers. However, there are no canonical Clock/BMAL binding sites within the functional 1.1 kb exorh promoter region identified by Asaoka and colleagues [5]. Further, a search of genomic sequences 5 kb upstream of the exorh start codon, all of the exorh introns, and the 751 bp of available exorh downstream sequence reveals only one canonical Clock/BMAL binding site, located at position −2040 within the exorh 5’ UTR (data not shown). Although it is possible that this site mediates transcription by Clock/BMAL, it seems unlikely as functional Clock/BMAL binding sites tend to be found in closely spaced clusters. For instance, functional Clock/BMAL sites within the aanat2 promoter are found within a 257 bp Pineal Restrictive Downstream Module (PRDM) that contains three Otx binding sites and two Clock/BMAL binding sites [1,2]. Similarly, the promoter for the Otx5 target gene reverb-alpha contains a 165 bp region upstream of the translation start site that contains two Clock/BMAL binding sites that are essential for Clock/BMAL mediated transcription in COS-1 cells [56]. One possibility is that there are additional Clock/BMAL sites further upstream or downstream of the coding sequence or non-canonical binding sites that we do not recognize. Alternatively, the lack of a clear Clock/BMAL regulatory unit could indicate that Per3 is acting to suppress exorh transcription indirectly through the regulation of another gene or through a mechanism that does not require Clock/BMAL (Figure 6).

In summary, our study makes three important advances in our understanding of pineal rhythms. Importantly, it defines the first in vivo role for Exorh protein in the regulation of transcription of its own gene and of aanat2. Second, it provides further evidence for the role of Otx5 as a major activator of rhythmic pineal genes. Finally, we establish a new role for Per3 as a key factor controlling the timing of exorh expression.

4. Experimental Procedures


Adult, embryonic, and larval fish were housed at 28.5°C in a 14:10 hr light/dark (LD) cycle. Fish strains included wildtype (WT) fish that were descendants of fish purchased from Scientific Hatcheries (Huntington Beach, CA, USA) or AB (Eugene, OR, USA), and exorh:GFP transgenic fish [5]. Embryos were obtained by natural matings and raised in aquatic system water containing 0.003% phenylthiocarbamide to inhibit the development of pigment. All embryos and larva were kept in temperature-controlled circadian incubators with LD cycles that matched parental lighting conditions. Position within the 24 circadian cycle was noted as Zeitgeber Time (ZT), with ZT0 corresponding to the time lights turned on and ZT14 corresponding to the time lights turned off. All procedures were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University, and were designed to minimize pain and discomfort.

MO injections

MO were manufactured by GeneTools, LLC (Philomath, OR, USA), with the following sequences: per3 MO, 5’-AGGAAAGCCGTCTCCCCCTGGCATT-3’; per3 MIS, 5’ AGcAAAcCCGTgTCCCgCTGcCATT-3’; exorh sp MO, 5’-TTGTAGTGTGCTCACCGCCGAGTGT-3’; exorh atg MO 5’-AGTTGGGTCCCTCCGTCCCGTTCAT-3’ standard control (ctl) MO, 5’-CCTCTTACCTCAGTTACAATTTATA-3’. The sequences for the otx5 MO and otx5 MIS were as previously described [20]. The exorh sp MO was designed using exorh genomic sequences obtained from the Sanger Center (Ensemble Gene ID ENSDARG00000046115).

1–2 cell stage WT embryos were injected with either 4 ng otx5 MO/otx5 MIS, 3 ng per3 MO/per3 MIS, 3 ng exorh sp MO/ctl MO or 1.5 ng exorh atg MO in 1× Danieau buffer [40] with a PLI-90 picoinjector (Harvard Apparatus, Holliston, Massachusetts, USA). The injected embryos were then raised and fixed in time courses as described in the figure legends.


Embryos were fixed in 4% paraformaldehyde in PBS at 4°C for at least 24 hours before being washed with PBS + 20% Tween 20. Samples were stored in 100% methanol at −20°C unless processed by in situ hybridization immediately. Transcripts of exorh [38], opn1lw1 [46], otx5 [20], per3 [18] and aanat2 [22] were detected by WISH as previously described [36]. Stained embryos were visualized using a Zeiss Axioplan2 imaging microscope. Digital images were captured with a Zeiss AxioCam HRm camera (Carl Zeiss MicoImaging Inc. Thornwood, NY, USA) in conjunction with OpenLab software (Scientific Software, Inc. Pleasanton, CA, USA) or with a SPOT RTke 7.4 slider Digital Camera along with SPOT Software version (Diagnostic Instruments Sterling Heights, MI, USA).

Quantification and Statistical Analysis

Digital images were converted to 8-bit grayscale using Adobe Photoshop CS2 version 9.0.1 (Apple software Cupertino, CA, USA). The optical density (OD) for a specified area was calculated from digital images of the pineal using ImageJ software version 1.36b (National Institutes of Health, Bethesda, MD, USA). The quantified area was the same for all samples within a single experiment.

For Figure 1B, the OD of the WISH signal was calculated for three pineal organs per time point. The values in each experiment were normalized by setting the highest OD reading to 100 and all other readings as a percentage thereof. Data were analyzed using one-way ANOVA with Tukey’s post hoc comparison of means and the Independent Two Sample Student’s t-Test in OriginLab 7.5 (OriginLab Corporation, Northampton, MA, USA).


Embryos at 3 dpf were processed for WISH with a probe for per3 or otx5. The brain was then sectioned by a sagittal cut through the midline using a standard disposable scalpel. For image capture, the sample was positioned so that the cut surface of the brain faced the camera.

mRNA injections

The mMessage Machine Kit (Ambion Inc. Austin, Tx, USA) was used to synthesize capped mRNA in vitro from the pCS2+otx5, pCS2+β-gal, and pCS2+ RFP plasmids [20,48,57]. Single blastomeres of 8–16 cell stage exorh:GFP embryos were injected with 0.5–1.5 nl of mRNA solution using a PLI-90 injector.

Supplementary Material


Supplementary Figure 1. Exorh protein is required for exorh transcription throughout the circadian cycle:

Embryos were injected with exorh atg or control MO, raised in a 14:10 hr LD cycle, fixed at the indicated time points, and processed for WISH using a probe for exorh. Exorh-deficient fish have lower levels of expression than control fish at all time points examined. All images are dorsal views, anterior to the top. Scale bar=20 µm.


The authors thank Drs. Kathleen Molyneaux, Greg Matera, Marge Sedensky, Phil Morgan, and Marnie Halpern for their helpful comments on the manuscript, Drs. David Klein (National Institutes of Health), Yoav Gothilf (Tel Aviv University), and Bernard and Christine Thisse (Institut de Génétique et Biologie Moléculaire et Celluaire) for generously sharing plasmids, Dr. Yoshitaka Fukada (The University of Tokyo) for generously providing the exorh (−1055):GFP transgenic line and the exorh cDNA, Dr. Mario Caccamo (Sanger Center) for advice on exorh genomic sequence, and Ms. Allisan Aquilina-Beck, Ms. Kristine Ilagan, and Mr. Brian Chen for their expert technical assistance. This work was supported in part by Research Grant No. 5-FY02-259 from the March of Dimes Birth Defects Foundation and Research Grant (J.O.L), No.T32 HD07104-29 Normal and Abnormal Development from the NIH/CHHD Institutional Pre-Doctoral Research Training Grant (L.X.P.), and Phi Beta Kappa Students Research Awards (O.P. and R.R.N.).


AANAT2Serotonin N-acetyl transferase 2
CrxCone rod homeobox
dpfdays post fertilization
ExorhExtra-ocular rhodopsin
GFPGreen Fluorescent Protein
hpfhours post fertilization
IRBPInterphotoreceptor retinoid binding protein
MISmissense morpholino
opn1lw1opsin 1(cone pigments) long-wave-sensitive, 1
Otx5Orthodenticle homeobox 5
Per3Period 3
RFPRed Fluorescent Protein
SCNsuprachiasmatic nucleus
WISHwhole mount RNA in situ hybridization
ZTzeitgeber time


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