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Keywords:

  • pineal gland;
  • epiphysis;
  • zebrafish;
  • circadian rhythm;
  • microarray

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The zebrafish pineal gland (epiphysis) is a site of melatonin production, contains photoreceptor cells, and functions as a circadian clock pace maker. Here, we have used microarray technology to study the zebrafish pineal transcriptome. Analysis of gene expression at three larval and two adult stages revealed a highly dynamic transcriptional profile, revealing many genes that are highly expressed in the zebrafish pineal gland. Statistical analysis of the data based on Gene Ontology annotation indicates that many transcription factors are highly expressed during larval stages, whereas genes dedicated to phototransduction are preferentially expressed in the adult. Furthermore, several genes were identified that exhibit day/night differences in expression. Among the multiple candidate genes suggested by these data, we note the identification of a tissue-specific form of the unc119 gene with a possible role in pineal development. Developmental Dynamics 238:1813–1826, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The pineal gland (epiphysis) is located at the dorsal edge of the diencephalon in the zebrafish. The conserved function of this organ in all vertebrates is the synthesis and secretion of melatonin, a hormone that regulates a variety of circadian and circannual physiological processes (Arendt,1995; Falcon,1999; Klein,2004). Melatonin levels are high at night and low during the day, as a consequence of regulated transcription and stability of serotonin-N-acetyltransferase (AANAT), the rate-determining enzyme of melatonin synthesis. In zebrafish and certain other nonmammalian vertebrates, the melatonin producing cells of the pineal gland are photoreceptors that can rhythmically produce melatonin for several days in isolation, reflecting the presence of an autonomous circadian clock pacemaker within these photoreceptor cells (Bernard et al.,1997; Begay et al.,1998; Falcon,1999). Therefore, the fish pineal photoreceptor cell is a valuable model system to study circadian function, photodetection, and melatonin production.

In addition, the zebrafish pineal gland is the first site where neurogenesis occurs, being apparent at approximately 24 hours post fertilization (hpf; Chitnis and Kuwada,1990; Wilson and Easter,1991). The existence of neuronal cells in the pineal gland which send projections to the brain makes this tissue more heterogeneous as compared to the pineal gland of mammals (Masai et al.,1997). The neuronal patterning surrounding the pineal gland is regulated by the homeobox transcription factor floating head (flh) and by masterblind (mbl), which encodes the negative regulator of wnt signaling Axin. Flh−/− zebrafish show reduced neuronal production in the pineal gland, whereas mutations in mbl increase the number of pineal neurons throughout the dorsal forebrain (Masai et al.,1997). Furthermore, the basic helix loop helix (bHLH) transcription factors achaete/scute homologue 1a (ascl1a) and neurogenin1 (ngn1) act downstream of flh to regulate neurogenesis (Cau and Wilson,2003). The molecular mechanisms of pineal gland development and function beyond these initial steps of neurogenesis have not been fully explored. Recently, pineal development and its relationship to brain asymmetry has received considerable attention (Gamse et al.,2003,2005; Halpern et al.,2003; Aizawa et al.,2005,2007; Hendricks and Jesuthasan,2007; Kuan et al.,2007a,b). Asymmetry depends on the laterality of the parapineal and is controlled by Nodal signaling (Concha et al.,2000,2003; Liang et al.,2000).

Gene profiling of the pineal gland of the chicken and rat have identified many genes that are highly expressed in the pineal gland, show night/day differences, or both (Humphries et al.,2002; Bailey et al.,2003,2008; Fukuhara and Tosini,2008). Here we report the results of global transcriptome analysis of the zebrafish pineal gland taken from day 3 larvae to adults. Many highly abundant transcripts that had not been previously reported to be present in this tissue have been identified. Furthermore, many highly expressed genes were found to dynamically change their expression levels during development. These data provide a broad basis for further molecular analysis of pineal gland development and physiology.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

As a first step in data analysis, all pineal gland data were averaged including data obtained during the day and night and at all five developmental stages; brain data were treated similarly. Probe sets were selected with the following criteria: P value ≤0.05 and pineal/brain signal ratio ≥ 5. Among the total 15,503 probe sets in the globally averaged data pool, 94 met these criteria. Of these, 43 probe sets have been annotated (http://www.affymetrix.com/analysis/index.affx), and nearly half of them (21 probe sets) correspond to genes known to be highly expressed in the pineal gland, including aanat2 (Gothilf et al.,1999), floating head (Talbot et al.,1995), extra-ocular rhodopsin (Mano et al.,1999), phosducin (accession number XM_677731), Crx (Liu et al.,2001), and otx5 (Gamse et al.,2002). Expression of GFP in the pineal gland of the transgenic fish used in this study was confirmed. These observations provide a first-level indication that our data effectively discriminate between genes that are differentially expressed in the pineal gland and the brain. Furthermore, principle component analysis of individual repeats indicated that the data are of high quality (not shown).

Genes Highly Expressed in the Pineal Gland Relative to Brain

Genes were considered to be highly expressed in the pineal gland relative to brain if the probability of a difference was ≤ 0.05. Setting the absolute difference at greater than threefold changes the number of selected genes (more accurately probe sets) shown in Table 1. The number of genes highly expressed in the pineal gland identified in this manner is much higher in adults than in larvae, possibly reflecting functional maturation of the tissue. Similar numbers of transcripts were enriched in the samples collected during the day and night. Probe sets selected at a threefold criterion in the three larval stages or the two adult stages partially overlapped, as shown in Figure 1. Approximately 20–35% of the transcripts detected at each stage were not detected at other stages, while approximately 60% of probe sets overlapped in RNA samples of 3 month and 1- to 2-year-old zebrafish. The total number of nonoverlapping probe sets at larval stages was 128 during the day and 150 at night, while 1,018 and 1,017 nonoverlapping probe sets were selected in adult day and night samples, respectively. Approximately 60% of probe sets identified as highly expressed in the larval pineal gland were also found to be enriched in adult tissue.

Table 1. Number of Genes Enriched in the Pineal Gland as Compared to Brain (P/B), Listed at Different Enrichment Ratiosa
 d3d5d103mo1-2 yr
  • a

    Samples were analyzed at midday and midnight at each developmental stage. The following criteria were used for the selection of probe sets. Average minimum signal ≥ 200; P value ≤0.05.

Day     
 P/B     
 ≥31006456751724
 ≥5503728385363
 ≥10211917155194
Night     
 P/B     
 ≥31027091878566
 ≥5533549432245
 ≥10231921191123
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Figure 1. Overlap of pineal-enriched genes at different stages. Venn diagrams of genes enriched in the pineal gland threefold or higher compared with the brain at different stages; larval and adult stages are shown separately. The total number of nonoverlapping enriched genes is 128 (larvae, day), 150 (larvae, night), 1,018 (adult, day), 1,017 (adult, night).

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We next compared the results of day and night analysis. Among the 128 genes highly expressed in the larval pineal gland relative to the brain at day and the 150 genes highly expressed at night, 83 were expressed both at day and night. Because more than 1,000 probe sets were selected as pineal enriched transcripts in adult samples under these criteria, we applied more stringent criteria to select genes. Increasing the required pineal/brain difference from three- to fivefold while maintaining all other conditions, the numbers of nonoverlapping probe sets highly expressed in adult (3 month and 1–2 years old) pineal glands were 322 and 365 at day and night, respectively; among these, 197 were expressed during both day and night.

The 50 annotated genes most highly enriched in the pineal gland in at least one of the stages studied are presented in Table 2 (larvae) and Table 3 (adult). More complete information on the genes selected by our criteria at larval and adult stages are listed in Supplementary Table S1 (3× enrichment, which is available online) and Supplementary Table S2 (5× enrichment). These lists include many more genes highly expressed in the pineal gland than previously identified. Many of these genes code for proteins involved in photoreceptor signal transduction pathways including G proteins and cGMP specific phosphodiesterase. We also identified a homolog of unc119, rbp4 (retinol binding protein 4), zic family members, and iroquois homeobox proteins as transcripts that are pineal enriched as compared to the brain. The unc119 homolog was studied further (see below).

Table 2. Top 50 Pineal Gland Enriched Genes at Larval Stagesa
 Probe setGene titleGene symbolP/B
  • a

    Values of P/B fold change for all six individual analyses (3d day, 5d day, 10d day, 3d night, 5d night, 10d night; see Supp. Table S1 for individual values) were averaged.

1Dr.352.1.S1Floating headflh66.4
2Dr.9835.1.S1Guanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 1gngt157.5
3AFFX-Dr-U43284-1_sGFPGFP47.7
4Dr.10292.1.S1Retinol binding protein 4, likerbp4l37.4
5Dr.9876.1.S1Guanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 2gngt230
6Dr.9829.1.S1Phosphodiesterase 6G, cGMP-specific, rod, gammapde6g26.5
7Dr.9853.1.A1Phosphodiesterase 6A, cGMP-specific, rod, alphapde6a22.5
8Dr.9908.1.A1Similar to ENSANGP00000004777/LOC557454 21.7
9Dr.12451.1.S1Retinal pigment epithelium-specific protein arpepa17.9
10Dr.5738.1.S1Similar to interphotoreceptor retinol-binding protein/LOC563355 17.7
11Dr.12451.2.A1Retinal pigment epithelium-specific protein arpepa15.2
12Dr.9899.1.S2Guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 1gnat112.7
13Dr.8142.1.S1Arylalkylamine N-acetyltransferaseaanat211.3
14Dr.9871.1.A1Recoverinrcv19.5
15Dr.8099.1.S1Extra-ocular rhodopsinexorh9.4
16Dr.12762.1.A1Phosducin 2/ similar to Pdc2 protein/LOC100007784pdc29
17Dr.12469.1.S1Arrestin 3, retinal (X-arrestin), likearr3l9
18Dr.19931.1.S1Tryptophan hydroxylase 1 (tryptophan 5-monooxygenase)tph18
19Dr.20586.1.A1Similar to agouti related protein 2/LOC796595 7.6
20Dr.15967.1.A1Tryptophan hydroxylase 1 (tryptophan 5-monooxygenase)tph17.4
21Dr.9845.2.A1ADP-ribosylation factor-like 3, like 2arl3l27.1
22Dr.9841.1.A1Phosphodiesterase 6C, cGMP-specific, cone, alpha primepde6c6.7
23Dr.11240.1.A1Similar to gefiltin/LOC555251 6.4
24Dr.14052.1.A1Tryptophan hydroxylase 2 (tryptophan 5-monooxygenase)tph26.3
25Dr.24898.1.S1Chromosome 20 open reading frame 149 homolog (human)c20orf1496.3
26Dr.11286.1.S1Phospholipase A1 member Apla1a6.2
27Dr.11085.1.A1Retinaldehyde binding protein 1, likerlbp1l6
28Dr.5167.1.A1Similar to lambda-recombinase-like protein/LOC100004795 6
29Dr.12902.1.A1Cytochrome P450, family 11, subfamily B, polypeptide 2cyp11b25.6
30Dr.9899.1.S1Guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 1gnat15.6
31Dr.10689.1.S1zic family member 2 (odd-paired homolog, Drosophila) bzic2b5.5
32Dr.6658.1.A1Ca2+-dependent activator protein for secretion 2cadps25.4
33Dr.14325.1.S1Cone-rod homeoboxcrx5.4
34Dr.11305.1.A1Guanylate kinase 1guk15
35Dr.12592.1.S1Guanylate cyclase activator 1Aguca1a4.9
36Dr.17145.1.S1Potassium channel tetramerization domain containing 12.1/similar to leftover/LOC796664kctd12.14.9
37Dr.16367.1.A1Similar to centrin/LOC795513 4.6
38Dr.9845.1.S1ADP-ribosylation factor-like 3, like 2arl3l24.5
39Dr.16724.1.A1Transcribed locus, strongly similar to NP_001076421.1 si:ch211-221n23.1 4.4
40Dr.26347.1.A1Pyrophosphatase (inorganic)pp4.3
41Dr.26319.1.A1Dopa decarboxylaseddc4.1
42Dr.284.2.A1_aOrthodenticle homolog 1otx14.1
43Dr.22887.1.A1Similar to zinc finger protein Zic6/LOC796374 4
44Dr.13970.1.S1ADP-ribosylation factor-like 4aarl4a4
45Dr.4807.1.S2zic family member 2 (odd-paired homolog, Drosophila), azic2a3.9
46Dr.12624.1.S1_aIroquois homeobox protein 1, birx1b3.9
47Dr.10724.1.S1Eomesodermin homolog aeomesa3.8
48Dr.1730.1.A1Similar to complement control protein factor I-B/LOC557557 3.6
49Dr.9881.2.A1Guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 2gnat23.6
50DrAffx.2.17.S1Gamma-aminobutyric acid (GABA) B receptor, 1gabbr13.5
Table 3. Top 50 Pineal Gland Enriched Genes in Adult Stagesa
 Probe set IDGene titleGene symbolP/B
  • a

    Adult stages (see Supp. Table S2 for individual values) were analyzed as described in Table 2.

1Dr.10292.1.S1Retinol binding protein 4, likerbp4l211.4
2Dr.12592.1.S1Guanylate cyclase activator 1Aguca1a203.5
3Dr.12469.1.S1Arrestin 3, retinal (X-arrestin), likearr3l176.7
4AFFX-Dr-U43284-1_sGFPGFP169.7
5Dr.12451.1.S1Retinal pigment epithelium-specific protein arpepa162.5
6Dr.9908.1.A1Similar to ENSANGP00000004777/LOC557454 154.6
7Dr.9853.1.A1Phosphodiesterase 6A, cGMP-specific, rod, alphapde6a130.3
8Dr.9841.1.A1Phosphodiesterase 6C, cGMP-specific, cone, alpha primepde6c128
9Dr.9871.1.A1Recoverinrcv1125.2
10Dr.11305.1.A1Guanylate kinase 1guk1119
11Dr.8099.1.S1Extra-ocular rhodopsinexorh110.8
12Dr.9899.1.S1Guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 1gnat1101.8
13Dr.5738.1.S1Similar to interphotoreceptor retinol-binding protein/LOC563355 94.7
14Dr.9899.1.S2Guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 1gnat192.2
15Dr.9876.1.S1Guanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 2gngt290.9
16Dr.9835.1.S1Guanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 1gngt190.9
17Dr.8071.1.S1Opsin 1 (cone pigments), long-wave-sensitive, 1opn1lw184.2
18Dr.12762.1.A1Phosducin 2/similar to Pdc2 protein/LOC100007784pdc279.3
19Dr.12451.2.A1Retinal pigment epithelium-specific protein arpepa78.1
20Dr.352.1.S1Floating headflh62.9
21Dr.14052.1.A1Tryptophan hydroxylase 2 (tryptophan 5-monooxygenase)tph258.5
22Dr.8142.1.S1Arylalkylamine N-acetyltransferaseaanat255.5
23Dr.24898.1.S1Chromosome 20 open reading frame 149 homolog (human)c20orf14949.5
24Dr.15426.1.S1Orthodenticle homolog 5otx547.8
25Dr.19931.1.S1Tryptophan hydroxylase 1 (tryptophan 5-monooxygenase)tph143.7
26Dr.11085.1.A1Retinaldehyde binding protein 1, likerlbp1l41.8
27Dr.1730.1.A1Similar to complement control protein factor I-B/LOC557557 40.1
28Dr.20586.1.A1Similar to agouti related protein 2/LOC796595 38.4
29Dr.25442.1.A1Elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 4elovl435.5
30Dr.15967.1.A1Tryptophan hydroxylase 1 (tryptophan 5-monooxygenase)tph133.4
31Dr.9845.2.A1ADP-ribosylation factor-like 3, like 2arl3l233.3
32Dr.2377.1.A1Keratin, type 1, gene 19dkrt1-19d31.9
33Dr.14325.1.S1Cone-rod homeoboxcrx31.9
34Dr.26319.1.A1Dopa decarboxylaseddc29
35Dr.9881.1.S1Guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 2gnat225.5
36Dr.20115.1.S1Cofilin 1 (non-muscle)cfl125.4
37Dr.2377.2.S1Keratin, type 1, gene 19dkrt1-19d24.4
38Dr.26347.1.A1Pyrophosphatase (inorganic)pp23.8
39Dr.9881.2.A1Guanine nucleotide binding protein (G protein), alpha transducing activity polypeptide 2gnat222.8
40Dr.4833.1.S1Annexin A1c/zgc:86853anxa1c21.7
41Dr.19801.1.A1Similar to fatty acid binding protein 1b/LOC795525 21.4
42Dr.994.1.S1Claudin bcldnb21.3
43Dr.24943.1.S1Similar to Desmoglein 2/LOC560026 21.3
44Dr.11283.1.A1Solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3, likeslc25a3l21.1
45Dr.25140.7.A1_aTumor-associated calcium signal transducer/zgc:110304/ protein LOC791868/similar to pan-epithelial glycoprotein/LOC100000093tacstd20.9
46Dr.6924.1.S1S-adenosylhomocysteine hydrolaseahcy20.4
47Dr.3499.3.A1Coronin, actin binding protein, 1Acoro1a19.1
48Dr.1434.1.S1Keratin 5krt518.5
49Dr.25556.1.S1Keratin 15krt1518
50Dr.9829.1.S1Phosphodiesterase 6G, cGMP-specific, rod, gammapde6g18

At least two habenula-specific genes, cadps2 and leftover, were also identified as pineal enriched genes (Table 2, #32 and #36, respectively). Because habenular nuclei are located very close to the pineal gland at larval stages, this may be due to contamination of the pineal sample with habenular tissue. As an independent approach to test the quality of the analysis, we picked a random series of unannotated clones and analyzed their expression pattern by in situ hybridization. Twelve of 19 clones analyzed showed enhanced expression in the pineal gland relative to the surrounding brain (Supp. Fig. S1, which is available online), while the remaining clones, whose expression levels were moderate to weak in the microarray analysis, were not detected by in situ hybridization (Supp. Fig. S1).

Analysis of Genes Highly Expressed in the Pineal Gland

To obtain functional insights into the categories of genes highly expressed in the pineal gland, Gene Ontology (GO) analysis was done using human homologs, as described in the Experimental Procedures section. Human homologs could be identified for 43–50% of zebrafish probe sets annotated for the Affymetrix Genechip. Approximately 50% of zebrafish probe sets are not well annotated (referred to as “transcribed loci” or “hypothetical proteins”) and were not assigned human homologues. Similar GO analysis done using the available zebrafish gene names was less informative (data not shown).

A summary of the GO analysis at Biological Process level 4 of genes highly expressed in the pineal gland is shown in Figure 2. For this analysis, we selected at threefold enrichment, nonredundant probe sets for the three larval stages and for the two adult stages, both at day and night (Fig. 1), and converted these groups of genes to their human homologs. The most highly represented GO terms are related to photoreception (e.g., “visual perception” and “detection of light stimulus”), both in larval and adult stages. Terms for neuronal development and function (“neurotransmitter metabolism,” “central nervous system development,” and “transmission of nerve impulse”), “nitrogen compound biosynthesis,” and “aromatic compound metabolism” were enriched at larval stages only, while GO terms related to programmed cell death and signal transduction (“intracellular signaling cascade” and “regulation of signal transduction”) were found only at adult stages (Fig. 2). An additional GO analysis at Molecular Function level 4 showed that the terms “retinal,” “retinol binding,” and “G-protein coupled photoreceptor activity” were found prominently in all four samples (Supp. Fig. S2); again these terms suggest enrichment of genes related to visual perception.

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Figure 2. GO term analysis of pineal-enriched genes at biological processes (BP) level 4. GO terms with P values < 0.1 (larvae) and < 0.05 (adult) were selected. Nonoverlapping genes were considered (Fig. 1). D, day, and N, night. The width of each slice indicates the number of genes in a given GO term. The numbers next to the colored squares indicate the GO terms, arranged by P values (small to large), corresponding to the numbers in parentheses in the table below. These P values indicate the probability of finding this GO term occupancy by chance, with a blank space indicating that the GO term was not enriched in the pineal gland. The color scheme starts at 12 o'clock and proceeds clockwise.

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Pathway analysis, as described in the Experimental Procedures section, identified “phototransduction pathway” as the most likely represented pathway in all larval and adult samples (Fig. 3). “Axonal guidance signaling” was also enriched in adult but less so in larval samples. Several signaling pathways were enriched only at adult stages, and among them “p53 signaling” was particularly interesting because of its involvement in programmed cell death which was found to be enriched in the GO analysis above (Fig. 2).

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Figure 3. Canonical pathways enriched in the pineal gland. Pathways enriched in all four samples (larvae day [D] and night [N], adult day [D] and night [N]) are shown at the top. The pathways shown at the bottom are enriched mainly in the adult pineal gland.

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These analyses indicate that pineal gene expression is enriched in classes of genes among which those involved in phototransduction are most prominent, consistent with the function of the fish pineal gland as a photoreceptor organ. Furthermore, significant differences were found between gene sets enriched in larval and adult pineal glands.

Transcriptome Analysis of Developmentally Regulated Genes

Developmental changes in pineal gene expression were identified by searching for genes that exhibited a ≥ threefold difference between the highest and lowest signal value among the five stages examined (3 days, 5 days, 10 days, 3 months, and 1–2 years), having a signal ≥ 200 in at least one stage, and P value ≤ 0.05. This analysis does not consider enrichment in the pineal relative to the brain. Using these criteria, 2,370 probe sets were selected from daytime pineal gland data as changing during development. Cluster analysis was performed with these probes, and the set was divided into four subclusters (Fig. 4). In broad terms, subsets A and B contain genes whose expression levels are low during larval stages but increased at adult stages, while subsets C and D show the opposite profile. GO analysis of clusters A and C was done, while subsets B and D contain too few genes for this analysis. Subsets A and C, which contained 1,191 and 733 zebrafish probe sets, respectively, were converted to 552 and 299 human homologs as indicated above. The results of GO analysis at Biological Process level 5 and Molecular Function level 3 are shown in Figure 5 and Supplementary Figure S3, respectively.

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Figure 4. Hierarchical clustering of genes whose expression levels change during the day in the pineal gland during development. Genes were selected using the following criteria: average maximum signal ≥200; lowest-to-highest signal ratio ≥ 3; P value ≤0.05. Low expression: green; high expression: red. Samples were grouped into four subsets, A–D. 1, 3 days; 2, 5 days; 3, 10 days; 4, 3 months; 5, 1–2 years.

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Figure 5. GO term analysis of subsets A and C of Figure 4 at biological processes (BP) level 5. GO terms which P values ≤0.05 were selected. The numbers next to the colored squares correspond to the numbers in the table below; see also legend to Figure 2.

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Similar analysis with the pineal gland “night” samples produced essentially similar results (Supp. Figs. S4, S5, S6).

In subset A, GO terms related to phototransduction were considerably enriched (Fig. 5; Supp. Figs. S3, S5, S6). Furthermore, terms related to cell death are also represented (Fig. 5; Supp. Fig. S5). These GO terms were found in genes enriched in the adult pineal gland, reflecting physiological function rather than development of the pineal gland. In contrast, terms related to transcription were highly enriched in subset C (Fig. 5; Supp. Figs. S3, S5, S6). Likewise, GO terms such as “brain development” and “neuron differentiation” were found in genes enriched at larval stages (Fig. 5; Supp. Fig. S5). These terms clearly reflect developmental changes that occur during these stages.

To illustrate the classes of genes that represent the different categories we chose GO terms that were most significantly represented in each subset. For each of these terms, we show the genes included as hierarchical clusters in Figures 6 (subset A) and 7 (subset C). It is apparent that groups of genes included in certain GO terms are co-regulated at different stages in the pineal gland.

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Figure 6. Clustering of genes in the selected GO terms shown in Figure 5, subset A. Each row of the heat map (see Fig. 4 for explanations) corresponds to one probe set. Affymetrix probe set ID numbers and gene symbols are shown on the left. 1, 3 days; 2, 5 days; 3, 10 days; 4, 3 months; 5, 1–2 years.

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Analysis of Genes That Exhibit Night/Day Expression Differences in the Pineal Gland

The mid-day and mid-night gene expression profiles of the pineal gland were compared at different developmental stages. Although this approach does not necessarily reflect circadian clock control of gene expression and is likely to miss changes that do not peak at midday or midnight, it provides a useful approach to characterizing daily changes in gene expression.

Probe sets were selected using the following criteria: P value ≤0.05 and night and day signal ratio ≥1.5 or 2 (Fig. 8). Approximately 250 genes were identified that showed at least ≥1.5-fold differences in expression between day and night in four of the five stages tested. There was no significant difference in the number of genes selected between larvae at d5 and d10 and adults at 3 months and 1–2 years, however, a higher number of genes exhibited day/night differences at larval stage d3. The biological meaning of this result remains to be elucidated.

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Figure 8. Number of genes highly expressed at day or night in the pineal gland. Samples were analyzed at each developmental stage separately. The following criteria were used for the selection of probe sets: Average minimum signal ≥100; P value ≤0.05. Dark gray: D/N≥1.5; black: D/N≥2; light gray: N/D≥1.5, white: N/D≥2.

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The probe sets showing day/night differences in at least four of the five developmental stages are listed in the Supplementary Table S3. aanat2 was selected as a gene highly expressed at night, as expected. Another gene highly expressed at night is bHLH domain containing class B, an inhibitor of the BMAL:CLOCK circadian transcription activator. The circadian clock gene per2 and rbp4 (retinal binding protein 4), which was also identified as highly expressed in the pineal gland compared with the brain, were selected as being highly expressed during the day. These results further strengthen the validity of the microarray analysis and the genes identified here.

unc119 Homolog Is Highly Enriched in the Pineal Gland

Unc119 homolog (Dr. 9908, Supp. Table S1) was identified as a gene of special interest because it is highly expressed in the pineal gland at both day and night compared with the brain at all stages we examined, and might play a role in pineal gland development and/or function. Because this is the third family member of unc119 found in the zebrafish, we refer to this gene as unc119c.

Unc119c shares 45.7% amino acid identity with the previously published zebrafish Unc119 (Manning,2004) and is clearly most diverged from Unc119 homologues in other species (data not shown). In situ hybridization experiments revealed that unc119c is expressed in the pineal gland at 72 hpf (Fig. 9A). The transcript is not found at the 20 somite stage or earlier (data not shown).

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Figure 9. unc119c and arl3l2 are co-expressed in the pineal gland and interact with each other. A,B: Spatial expression of zebrafish unc119c (A) and arl3l2 (B). Both panels are shown as dorsal views of 3 dpf larvae. In situ hybridization was carried out as described (Toyama and Dawid,1997). C: Co-immunoprecipitation of zebrafish Unc119c and Arl3l2 transfected into HEK293 cells. The antibody used for precipitation is indicated by IP, and the antibodies used for blotting are shown on the right side of the panels. Myc-tagged Arl3l2 co-precipitated with Flag-Unc119c (white arrowhead in the top panel, shown above immunoglobulin light chain.), but Myc-tagged nucleolin (Myc-Nucleo) did not. Black arrowheads indicate Myc-tagged Arl3l2 and Nucleolin (used as negative control) in the middle panel.

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Recently, ADP-ribosylation factor-like protein 2 (ARL2) has been identified as an interacting protein of a human homologue of UNC119, HRG4 (Kobayashi et al.,2003). Of interest, members of the arl gene family, arl3l1 (ADP-ribosylation factor-like protein 3 like 1) and arl3l2, were highly enriched in the pineal gland relative to brain in our microarray data. arl3l2 is expressed in the zebrafish pineal gland, as visualized by in situ hybridization (Fig. 9B), suggesting a possible interaction between Unc119c and Arl3l2.

To test the possibility that Unc119c and Arl312 interact, we performed co-immunoprecipitation studies with FLAG-tagged Unc119c and myc-tagged zebrafish Arl3l2 in cultured cells. Arl3l2 specifically interacted with Unc119c (Fig. 9C). This suggests that these two proteins may be part of a common functional pathway in the zebrafish pineal gland.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Genes Highly Expressed in the Pineal Gland

In this study, we have systematically examined the gene expression profiles in the larval and adult zebrafish pineal gland. To identify genes whose expression is enriched in the pineal gland, we used brain tissue without pineal gland and without eyes as reference. Eyes were removed from control samples because the zebrafish pineal gland is a photoreceptive organ and, therefore, similar molecular pathways may be active in eyes and pineal gland.

While there is significant overlap between genes highly expressed in the pineal gland at larval and adult stages, we considered the two groups of data separately for GO analysis. This approach revealed the enrichment of developmentally specific GO terms which would be obscured by combining all the data. As result, we found that GO terms for neuronal development and function were specifically enriched at larval stages.

Throughout this microarray analysis, we obtained evidence for many genes that are highly expressed in the pineal gland relative to brain. At present, nearly half of the probe sets are not well annotated, being referred to as “transcribed locus” or “hypothetical protein.” Future expansion of the EST database and completion of the zebrafish genome will promote the analysis of these genes. Nevertheless, we generated a list of many annotated genes which have not been reported previously as expressed in the pineal gland. Individual analysis of some of these genes, and of genes that may be annotated in the future, offer the opportunity to identify new players in pineal development and function. Some of these newly identified pineal genes may relate to the establishment of asymmetry in the pineal and surrounding epithalamic region (Harris et al.,1996; Concha and Wilson,2001; Gamse et al.,2003; Snelson et al.,2008). This proposal is supported by the finding that otx5, which is enriched in the pineal gland, also exhibits asymmetric expression in the zebrafish epithalamus.

The finding of a highly expressed transcript in the pineal gland provides reason to characterize unannotated genes because such highly expressed genes are likely to be functionally important. In addition to the unc119 homologue (see below), another interesting novel gene is the zebrafish homolog of agrp which corresponds to probe set Dr. 20586.1.A1 (Supp. Table S1). AgRP is a key hypothalamic regulator of ingestive behavior in mammals and zebrafish (Song et al.,2003). An immunocytochemical study in zebrafish using an antibody against human AgRP revealed unexpected AgRP immunoreactivity in the pineal gland, leading to the suggestion that this was due to the expression of the gene in the pineal gland termed AgRP2 (Forlano and Cone,2007). Here, we independently reached the same conclusion based on our microarray analysis. The pineal-enhanced expression of the AgRP2 homolog suggests a possible connection between the circadian oscillator in the zebrafish pineal to ingestive behavior, an attractive direction for further research.

Previous Microarray Studies on Gene Expression in the Vertebrate Pineal Gland

Three groups have previously published results of microarray analysis of the rat pineal gland (Humphries et al.,2002; Bailey et al.,2008; Fukuhara and Tosini,2008). Fukuhara et al. found that approximately 2% of a total of 8,000 genes on the microarray showed rhythmic expression in the pineal gland, consistent with the findings by Humphries et al. (approximately 3% out of 1,176 genes). Bailey et al. found that approximately 4% of the genes (∼600 of ∼13, 000) exhibit greater than twofold change on a night/day basis. Our data identified approximately 500 genes which are up-regulated at day or night in the adult zebrafish pineal gland (approximately 3% of all genes on the microarray). Our finding agrees with previous observations (Humphries et al.,2002; Bailey et al.,2008; Fukuhara and Tosini,2008) that only a limited number of genes exhibited day/night differences in their expression level.

All previous analyses were done with adult rat pineal gland and found that more genes were up-regulated at night as compared to those up-regulated during the day (47 vs. 13; Fukuhara et al.,2008). Likewise, Bailey et al. found approximately twofold more genes that were elevated at night (Bailey et al.,2008). In contrast, we found similar numbers of genes up-regulated at day and at night. Furthermore, we identified many more genes showing day/night differences in expression, which had not been reported previously. Only few genes previously reported to display a diurnal rhythm of expression were identified in our analysis. One of the genes characterized by Humphries et al. (2002) as a nocturnal up-regulated gene, Id-1 (inhibitor of DNA binding and differentiation), did not show significant day/night changes in its expression in the zebrafish pineal gland. These differences may be caused by species differences or by incomplete annotation of the zebrafish microarray.

Possible Functional Interaction Between Unc119c and Arl3l2

The unc119 gene is a new class of neural gene that shares conserved sequences in all metazoans examined. Although this gene is highly conserved from worms to human, its expression pattern shows two extremes. In invertebrates (C. elegans and Drosophila), this gene is expressed throughout the nervous system, and unc119 knockout causes damage to neurons widely distributed in the nervous system (Maduro et al.,2000; Knobel et al.,2001). In mammals (human, mouse, rat), this gene was originally identified as HRG4 (human retinal gene 4) (Higashide et al.,1996), a gene that is expressed specifically in the photoreceptor synapse. A truncation mutation of HRG4 is associated with late-onset cone-rod dystrophy in humans, and transgenic mice containing the same mutation, develop late-onset retinal degeneration (Kobayashi,2000). HRG4 knockout in the mouse causes severe damage to the retina (Ishiba et al.,2007). In zebrafish, one unc119 homolog has been identified (unc119) with an expression pattern similar to that seen in invertebrates, i.e., expression throughout the central nervous system; its knock down results in disorganized neural architecture (Manning et al.,2004). A second unc119 homolog (unc119b) was found in the zebrafish genome but its expression pattern was not characterized (Manning et al.,2004). Here, we show the existence of a third unc119 homolog (unc119c) that exhibits enhanced expression in the pineal gland.

Although the function of UNC119 is not clearly understood, the recent finding that ARL2 (ADP-ribosylation factor-like protein 2) interacts with HRG4 suggests a possible function for UNC119 in the retina. ARL2 is a guanine nucleotide binding protein and may play a role in microtubule assembly (Radcliffe et al.,2000). ARL2 interacts with BART (binder-of-ARL2), enabling it to enter mitochondria and bind ANT-1 (adenine nucleotide transporter), which is thought to be involved in apoptosis (Mori et al.,2006). Therefore, a truncation mutation of HRG4 may lead to mitochondrial ANT-1-mediated retinal degeneration by apoptosis through ARL2.

Here, we demonstrate the coexpression of unc119c and arl3l2, one of the closest homologs of arl2, as well as the physical interaction between Unc119c and Arl3l2. To our knowledge, this is the first evidence suggesting an Unc119-Arl interaction in the pineal gland. Recently, Veltel et al. reported the formation of a ternary complex between Arl3, its GAP RP2 (retinitis pigmentosa 2), and HRG4 (Veltel et al.,2008). Also, mouse Unc119 interacts with the synaptic ribbon specific protein RIBEYE at photoreceptor ribbon synapses (Alpadi et al.,2008). The previously reported functions for UNC119 are all related to its expression in the retina. However, Bailey et al. also identified Unc119 as a highly expressed gene in the pineal gland and the retina relative to other tissues (Bailey et al.,2008), and our findings indicate that a distinct tissue-specific Unc119 homolog may play a role in photoreceptor cell function in the pineal gland. The high expression of Unc119 in photoreceptors might be a conserved feature in vertebrates, possibly based on similar functions. Whether the existence of an additional unc119 homolog is a unique feature of the zebrafish is not known, but it should be remembered that the zebrafish pineal gland is located superficially and contains photoreceptor cells, in contrast to the situation in higher vertebrates. Zebrafish may, therefore, have gained an additional unc119 gene that is functional outside of the retina.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Collection of Larvae and Adult Pineal Glands and RNA Preparation

Adults and larvae were kept under a 14-hr-light/10-hr-dark cycle. Pineal glands were isolated manually using a fluorescence dissection microscope, guided by green fluorescent protein (GFP) fluorescence, from larval (3 days, 5 days, and 10 days) and adult (3 month and 1–2 years) transgenic zebrafish in which expression of the GFP gene is driven by the aanat2 promoter (Gothilf et al.,2002). For comparison, brain tissue from which the pineal gland and eyes had been removed was also collected (referred to as “brain” from here onward). The tissues were collected directly in QIAzol (Qiagen). Five to nine adult or 10 to 38 larval pineal glands, and 2 to 3 adult or 2 to 5 larval brains were pooled to yield one sample. Three to five samples were collected at midday and midnight at each developmental stage.

Total RNA was prepared using the RNeasy Lipid Tissue Mini Kit (Qiagen) and biotin-labeled cDNA was generated using the Ovation Biotin system kit (NeuGen)

Microarray Analysis and Data Processing

The Affymetrix GeneChip Zebrafish Genome Array was hybridized and processed using the standard Affymetrix protocol. Altogether, we collected 20 types of samples: five time points (3 days, 5 days, 10 days, 3 months, and 1–2 years), two organs (pineal gland and brain), and two sampling times (day and night). For each type of sample, tissue was obtained and processed three to five times. After hybridization, microarray chips were scanned, quantitated, and normalized by GCOS (Affymetrix). All data were submitted to the NCBI GEO database as series GSE13371. There are 15,617 probe sets on a zebrafish Affymetrix microarray chip, including 114 hybridization controls; data for the remaining 15,503 probe sets were subjected to further statistical using JMP, the statistical Discovery Software (http://www.jmp.com/). Unless otherwise indicated, three to five replicates for each sample type were averaged, and probe sets showing differences with a P value smaller than 0.05 were considered further.

Gene Ontology (GO) Analysis

To take advantage of the more complete annotation of the human genome, we converted lists of selected zebrafish genes to those of their human homologs, using the WEB site of the Zebrafish Gene and Microarray Annotation Project (ZGMAP) (Children's Hospital Boston, http://134.174.23.160/zfACA/hash/cumulative_expanded.aspx). GO term analysis was performed using the DAVID Bioinformatics Resources, NIAID/NIH, with the default level of P < 0.05 to select genes to be included, unless noted otherwise.

Pathway Analysis

Ingenuity Pathways Analysis software (http://www.ingenuity.com/products/pathways_analysis.html) (ver.5) was used to identify canonical pathways most likely to be active in the pineal gland. Selected zebrafish genes were converted to a list of human genes as indicated above before this analysis.

Immunoprecipitation

HEK-293 cells were transfected with pcDNA3Unc119c-Flag, pcDNA3Arl3l2-Myc, and pcDNA3Nucleolin-Myc. Proteins were extracted and precipitated with anti-Flag antibodies (Sigma, F3165) coupled to anti-mouse IgG agarose beads (Sigma, A6531).

Proteins were detected with anti-Myc (Sigma, C6594) or anti-Flag (Sigma, F3165) antibodies.7

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Figure 7. Clustering of genes in the selected GO terms shown in Figure 5, subset C. Each row of the heat map corresponds to one probe set. Affymetrix probe set ID numbers and gene symbols are shown on the left. 1, 3 days; 2, 5 days; 3, 10 days; 4, 3 months; 5, 1–2 years.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Mark Rath for excellent technical assistance. This work was partially supported by a grant from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel, and by the Intramural Research Program of the NICHD, NIH.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Additional Supporting information may be found in the online version of this article.

FilenameFormatSizeDescription
DVDY_21988_sm_SuppFigS1.tif7000KSupp. Fig. S1. Spatial expression pattern of selected pineal enriched genes in day 3 zebrafish larvae. In situ hybridization was carried out as described (Toyama and Dawid, 1997). The Affymetrix probe set ID numbers corresponding to the different clones are indicated. A, Dr.9901.1.S1; B, Dr.6401.1.S1; C, Dr.11336.1.A1; D, Dr.9852.1.A1; E and F, Dr.12466.1.A1. F, eyes are removed. All panels show lateral views.
DVDY_21988_sm_SuppFigS2.tif3412KSupp. Fig. S2. GO term analysis of pineal enriched genes selected in Figure 1 at molecular function (MF) level 4. GO terms with P values ≤ 0.1 were selected. Each pie chart represents the results of a different stage (larval or adult) and time (day or night). Each slice indicates the number of genes in a given GO term. The numbers next to the colored squares indicate the order of GO terms arranged by P values (small to large), and correspond to the numbers in parentheses in the table below. The table lists the P values of GO terms in the left column. No entry indicates that the GO term was not enriched in the pineal gland at the given category.
DVDY_21988_sm_SuppFigS3.tif8351KSupp. Fig. S3. GO term analysis of subsets A and C shown in Figure 4 at molecular function (MF) level 3. GO terms with P values ≤ 0.1 were selected. See legend of Supplementary Figure S2 for further explanations.
DVDY_21988_sm_SuppFigS4.tif1213KSupp. Fig. S4. Hierarchical clustering of genes whose expression levels change at night in the pineal gland during development. Genes were selected using the following criteria: average maximum signal ≥200; lowest and highest signal ratio ≥ 3; P value ≤0.05. Low expression, green; high expression, red. Samples were grouped into four subsets, A-D. 1, 3 days; 2, 5 days; 3, 10 days; 4, 3 months; 5, 1-2 years.
DVDY_21988_sm_SuppFigS5.tif9346KSupp. Fig. S5. GO term analysis of subsets A and C shown in Supplementary Figure S4 at biological processes (BP) level 5. See legend of Supplementary Figure S2 for further explanations.
DVDY_21988_sm_SuppFigS6.tif7050KSupp. Fig. S6. GO term analysis of subset A and C shown in Supplementary Figure S4 at molecular function (MF) level 3. See legend of Supplementary Figure S2 for further explanations.
DVDY_21988_sm_SuppTablesS1-S3.xls165KSupp. Table S1. Genes enriched in the larval pineal gland. All genes selected from larval stages in Fig.1 (day non-overlap total: 128; night non-overlap total: 150) are listed. D, genes only in day sample; N, genes only in night sample; DN, genes in both day and night sample. Supp. Table S2. Genes enriched in the adult pineal gland. All genes selected from adult stages in Fig.1 (day non-overlap total: 1018; night non-overlap total: 1017) are listed. D, genes only in day sample; N, genes only in night sample; DN, genes in both day and night sample. Supp. Table S3. Genes highly expressed at night (A) or day (B) in the pineal gland. Samples were analyzed at each developmental stage separately with average minimum signal ≥100 and P value ≤0.05. Probe sets whose fold change between day and night are higher than 1.5 at least in four out of five developmental stages (d3, d5, d10, 3mo, and 1-2 yr) are listed.

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