Transcriptional Profiling of the Chick Pineal Gland, a Photoreceptive Circadian Oscillator and Pacemaker

Michael J. Bailey, Phillip D. Beremand, Rick Hammer, Deborah Bell-Pedersen, Terry L. Thomas and Vincent M. Cassone

Center for Biological Clocks Research (M.J.B., P.D.B., R.H., D.B.P., T.L.T., V.M.C.), Department of Biology (M.J.B., P.D.B., R.H., D.B.P., T.L.T., V.M.C.), Texas A&M University, College Station, Texas 77843-3258

Address all correspondence and requests for reprints to: Vincent Cassone, Department of Biology, Texas A&M University, College Station, Texas 77843-3258. E-mail: vmc{at}mail.bio.tamu.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Materials and Methods
 REFERENCES
 
The avian pineal gland contains both circadian oscillators and photoreceptors to produce rhythms in biosynthesis of the hormone melatonin in vivo and in vitro. The molecular mechanisms for melatonin biosynthesis are largely understood, but the mechanisms driving the rhythm itself or the photoreceptive processes that entrain the rhythm are unknown. We have produced cDNA microarrays of pineal gland transcripts under light-dark and constant darkness conditions. Rhythmic transcripts were classified according to function, representing diverse functional groups, including phototransduction pathways, transcription/translation factors, ion channel proteins, cell signaling molecules, and immune function genes. These were also organized relative to time of day mRNA abundance in light-dark and constant darkness. The transcriptional profile of the chick pineal gland reveals a more complex form of gene regulation than one might expect from a gland whose sole apparent function is the rhythmic biosynthesis of melatonin. The mRNAs encoding melatonin biosynthesis are rhythmic as are many orthologs of mammalian "clock genes." However, the oscillation of phototransductive, immune, stress response, hormone binding, and other important processes in the transcriptome of the pineal gland, raises new questions regarding the role of the pineal gland in circadian rhythm generation, organization, and avian physiology.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Materials and Methods
 REFERENCES
 
THE AVIAN PINEAL gland is a critical component of birds’ biological clocks and is an important model system for the study of circadian rhythm generation in general. The gland is important for overt circadian organization because surgical removal of the pineal gland disrupts and/or abolishes circadian rhythms in several species of birds (1, 2, 3, 4, 5). Further, the avian pineal gland contains both circadian oscillators and pacemaker(s) to drive circadian rhythms in the biosynthesis of the indoleamine hormone melatonin and photoreceptors to synchronize that rhythm to environmental lighting (6, 7). When placed in organ and/or cell culture, the pineal glands of several avian species express at least 4 circadian cycles of melatonin biosynthesis under constant conditions of continuous darkness or dim red light (8, 9, 10). Further, chick pineal glands in vitro respond to environmental light in at least three mutually exclusive ways: 1) phase-shift of the circadian cycle; 2) acute suppression of melatonin biosynthesis and release; and 3) decrease in rhythm damping (increase in amplitude) (8).

The cellular sites of the oscillatory and biosynthetic mechanism(s) generating melatonin rhythms within the gland reside in a single cell type, the pinealocyte (11), and the molecular mechanisms for the biosynthesis of melatonin within those pinealocytes have been thoroughly worked out (Fig. 1AGo) (12, 13, 14). The amino acid tryptophan is taken up and converted to 5-hydroxytryptophan (5HTP) by tryptophan hydroxylase (TrH; E.C. 1.14.16.4). Then, aromatic amino acid decarboxylase (AADC; E.C.4.1.1.28) converts 5HTP to serotonin (5HT). During the night, 5HT is converted to N-acetylserotonin (NAS) by arylalkylamine-N-acetyltransferase (AANAT; E.C. 2.3.1.87). NAS is the primary substrate for hydroxyindole-O-methyltransferase (HIOMT; E.C.2.1.1.4), which converts NAS to melatonin. The details of the molecular regulation of rhythmic melatonin biosynthesis vary among species (14). In the chick, TrH, AANAT, and HIOMT are regulated on a circadian basis by both transcriptional and by posttranscriptional mechanisms, including proteosomal proteolysis (12, 13, 15).



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Fig. 1. The Melatonin Biosynthesis Pathway and Simplified Transcriptional/Translational Feedback Model of Circadian Rhythm Generation in Mammals

A, Model depicting the melatonin biosynthesis pathway. The amino acid tryptophan (TrP) is taken up by the cell and converted to 5HTP by tryptophan hydroxylase (TrH; E. C. 1.14.16.4). Then, dopa decarboxylase (AADC; E. C.4.1.1.28) converts 5HTP to 5HT. During the night, 5HT is converted to NAS by AANAT (E. C. 2.3.1.87). NAS is the primary substrate for HIOMT (E. C.2.1.1.4), which converts NAS to melatonin. B, Simplified version of the interlocking transcriptional/translational feedback loops organized to control the generation of circadian rhythms. It consists of "negative elements" and "positive elements" in both flies and mammals. The negative elements in mammals comprise the period genes (green box) and cryptochromes (red box). These genes are transcribed in response to the dimerization of the positive elements, which include clock (orange oval) and the Bmals (blue oval), and their subsequent binding to an E-box in the promoter regions of several genes. The negative elements in turn are translated (red, green ovals), oligomerize in the cytoplasm and re-enter the nucleus to inhibit their and other genes’ transcription.

 
As stated above, the chick pineal gland is photoreceptive and responds to light in several ways. Several opsin-based photopigments and two cryptochromes have been isolated and characterized from this gland (16, 17, 18). Further, many of the known opsin-based signal transduction mechanisms are present in the gland (19, 20). However, the roles these putative photopigments play in each of these light-based processes or the second messenger systems underlying their role are not known.

In spite of the fact that much is known about pineal physiology, the molecular mechanisms by which melatonin is synthesized and the identity of photopigments and their signal transduction cascades, the molecular mechanisms involved in the circadian rhythm generation itself, and the coupling of this rhythm to pineal clock output are completely unknown. Analysis of the molecular mechanisms responsible for circadian rhythm generation in other model systems has advanced rapidly. Initiated by the discovery of the period gene in Drosophila (21), the molecular clock model has now expanded into a network of clock genes that form interlocking transcriptional/translational feedback loops of negative elements and positive elements (Fig. 1BGo) (22, 23, 24, 25). The negative elements in mammals comprise the period genes (per1-3) and cryptochromes (cry1, 2). These are transcribed in response to the dimerization of the positive elements, which include clock and the Bmals (Bmal1, 2), and their subsequent binding to E-boxes in the promoter regions of several genes. The negative elements in turn are translated, oligomerized, and then re-enter the nucleus where they inhibit their and other genes’ transcription.

Although several studies have used high-density oligo and cDNA microarrays with the goal of uncovering genes involved in the regulation of clock function and elements under clock control (26, 27, 28, 29, 30, 31, 32), none have comprised arrays derived from cDNAs isolated from a specialized clock tissue itself and none have focused on the chick pineal gland, a unique model system for the study of biological clocks. Our study utilizes the uniqueness of the pineal gland model and cDNA microarray technology to identify candidate molecular components involved in the pineal clock, including core oscillatory elements as well as components of the input pathway (photopigments) and outputs (clock-controlled genes). We validated the transcriptional profile generated from the pineal cDNA microarrays via northern and in situ analysis. These together have identified several interesting candidate pineal clock components that further our understanding of circadian biology and avian physiology.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Materials and Methods
 REFERENCES
 
Chick Pineal Expressed Sequence Tag (EST) Analysis
To optimize our chances of identifying important rhythmic transcripts in the chick pineal that would be under-represented in public databases, we began our analysis by creating a pineal EST database accessible through our Center for Biological Clocks homepage (http://www.tamu.edu/clocks/index.html) and used it as a source for generating high-density microarrays. The clones used for this database were chosen at random from two pineal cDNA libraries we constructed. One library was prepared from cDNA derived from RNA isolated from pineal glands harvested at midday [Zeitgeber time (ZT)-6], the other from cDNA generated from RNA obtained from pineal glands harvested at midnight (ZT-18).

We conducted one-pass sequence analysis from the 5' end of approximately 10,000 cDNAs. Approximately 5000 ESTs were sequenced from the ZT-6 and ZT-18 libraries, respectively. Sequences were filtered, trimmed and analyzed on a local basic local alignment and search tool (BLAST) server to assign potential gene IDs to the ESTs (33). A total of 9867 sequences were submitted for BLAST analysis; of these, 54% (n = 5284) had a BLAST hit. We have used several approaches to estimate the prevalence distribution and complexity of the pineal libraries. For example, contig assembly using phrap (http://www.phrap.org) suggests that only about 20% of the cDNAs in the library occurs at least twice. However, this is likely an underestimate. A more realistic estimate was obtained by determining the frequency at which the same annotated genes occurred in the library. Here, roughly 50% of the ESTs with a BLAST hit occurred two or more times. However, 75% of the cDNAs are represented five or fewer times, indicating the chick pineal mRNA population is complex. It is noteworthy that only 20 genes represent 8% of the cDNAs in the library. The most abundant is transthyretin, representing 2.8% of all cDNAs sequenced in the chick pineal database.

The microarrays used in these experiments were produced when the database contained approximately 8,000 entries. Currently, approximately 10,000 entries have been made in the chick pineal database.

Transcriptome Analysis
Using our method of hybridization and analysis, consistent, very high signal to noise ratios were obtained. Of the approximately 8000 pineal cDNAs represented on the microarray, 1797 oscillate with 2-fold amplitude or greater change in a light-dark (LD) cycle as indicated via a gene tree (Fig. 2AGo). The most abundant transcripts that were oscillating rhythmically in LD were transthyretin precursor (prealbumin; n = 184), cystatin (n = 34), HIOMT (n = 13), glyceraldehyde-3-phosphate dehydrogenase (G3PDH; n = 12), group III secreted phospholipase C (n = 9), actin (n = 7), AANAT (n = 6), and TrH (n = 5). Upon correction and removal of transcripts for redundancy (n = 518) and unknown/unclassified transcripts (n = 902) this data set is reduced to 377 unique classified genes (published as supplemental data on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org; see supplemental Table 1Go).



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Fig. 2. Gene Tree of Pineal Gland Transcripts Oscillating in a LD and DD Cycle

A, Pineal transcripts rhythmically expressed under LD conditions were organized into a gene tree to visualize transcript abundance patterns (Genespring). Increased transcript abundance is represented by red fluorescence, decreased by green, and nonchanging transcript abundance by the color yellow. All fluorescence values are relative to respective values obtained at ZT-18. Of the approximately 8000 pineal ESTs present on the microarray 1797 oscillate with at least a 2-fold amplitude change in a light dark cycle. This data set includes 382 unique classified genes; while 902 transcripts had no significant BLAST score. B, Pineal transcripts rhythmically expressed under DD conditions were also organized into a gene tree as in panel A. The number of rhythmic pineal transcripts observed was 682, with 128 of these transcripts being unique classified genes, whereas 254 transcripts exhibited no significant BLAST score.

 

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Table 1. Conserved Clock Controlled Elements

 
In constant darkness (DD), the number of rhythmic pineal transcripts observed was 682 as indicated via a gene tree (Fig. 2BGo). The most abundant cDNAs on the microarray that were oscillating rhythmically in both LD and DD were again transthyretin (n = 118), HIOMT (n = 12), cystatin (n = 7), actin (n = 5), G3PDH (n = 5), and TrH (n = 5). AANAT was expressed 3 times. Upon correction and removal of transcripts for redundancy (n = 302) and unknown/unclassified transcripts (n = 254), this data set is narrowed to 126 unique classified genes (see supplemental data Table 2).

The small percentage of genes rhythmically expressed in DD does not necessarily mean that those that do not meet our 2.0-fold criterion are light regulated genes. In fact, when the rigor of this 2.0-fold criterion is reduced to 1.5-fold, 291 of the 377 genes that met the 2.0-fold criterion in LD met the 1.5-fold criterion in DD, 77% of the rhythmic genes. These additional genes are listed in supplemental Table 3. Of the 86 genes that do not meet the 1.5-fold criterion, no single functional group stands out as being only light regulated. Notably, all of the photopigment-associated genes meet all criteria in LD and DD and are likely circadian-clock regulated.

The fact that the largest single category of genes that were rhythmic was ESTs that had not returned a significant BLAST hit should not be construed as there being a significant pineal specific transcriptome because our sequencing was only one pass from the 5' ends. Thus, many of the unknown genes may be identified with additional sequencing. We have recently been able to place some of these genes into contigs. Preliminary BLAST analysis of the contig consensus has allowed tentative identification of some of the unknowns. Of the 254 unknown rhythmic cDNAs under both LD and DD, about 21 appear to be additional transthyretin genes, four are likely TrH, three match cystatin, two with AANAT, and one each with EURL, G3PDH, purpurin precursor and CATRO cytochrome P450. At least seven fall into a single contig that has no BLAST match.

It was not surprising to see components of the melatonin biosynthesis pathway among the most abundant rhythmic transcripts in the pineal gland. The pineal gland is best known for its production of this hormone. However, it was surprising to observe the very large number of transcripts corresponding to transthyretin, which has been primarily associated with the choroid plexus. Because the pineal gland is connected to choroid plexus in many species, including chickens, it is possible that some choroid tissue may have been included in our dissections. Although the method used during pineal dissection excluded choroid tissue, it was possible some choroid cells reside within the pineal parenchyma. Indeed, in situ hybridization (ISH) mRNA transthyretin in chick pineal revealed that the gland is surrounded by very intense transthyretin signal and infiltrated in the inferior portions of the gland by less intense transthyretin mRNA (see Fig. 9DGo), suggesting that in the chick pineal gland, choroid tissue is intimately associated with the gland. Transthyretin is linked to thyroid hormone binding, as a vitamin A carrier via an interaction with retinol binding protein, lipid transport, and as a marker of nutritional fitness (34). Preliminary data from our laboratory indicate that transthyretin binds melatonin with reasonably high affinity (Sacchettini, J., and V. M. Cassone, unpublished). Perhaps, transthyretin binds melatonin in a heretofore-unrecognized release mechanism for melatonin.



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Fig. 9. ISHs of Transcripts in the Chick Pineal

A, Positive control indicating per2 mRNA expression. B, cry1. C, B cell associated protein 2. D, Transthyretin. Black arrows indicate pineal gland position.

 
Functional Categories of Rhythmically Expressed Transcripts
Oscillating pineal gland transcripts in LD and DD were classified according to proposed function, revealing a wide variety of potential biological activities. First, the entire melatonin generating enzymatic cascade was represented, as were many chick orthologs associated with circadian clock function (clock genes). Further, cluster analysis revealed transcripts in several broad categories that were also rhythmically expressed in the chick pineal gland. Categories examined include transcription factors, ribosomal and translation factors, hormones and growth factors, carrier and transport proteins, components of cell signaling/adhesion, metabolic components, retinal and phototransduction elements, and stress response and host defense elements. The percentage of rhythmic transcripts represented in each category is also given (Fig. 3Go, A and B). The stress response cluster includes immune function genes, which under LD and DD had the highest number of oscillating pineal transcripts, 40 and 16, respectively. These functional clusters as well as others are also indicated in supplemental Tables 1Go and 2.



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Fig. 3. Clustering of Oscillating Pineal Transcripts via Proposed Function

A, Rhythmically expressed transcripts under LD conditions were also clustered according to proposed function into a pie chart, revealing a wide variety of applications. First, the entire melatonin generating enzymatic cascade was represented on the array, as were many chick orthologs associated with circadian clock function (clock genes). In addition, cluster analysis revealed transcripts in several broad categories were also rhythmically expressed in the chick pineal gland. The percentage of rhythmic transcripts next each proposed functional category name is also given. B, This functional categorization method was also applied to transcripts oscillating under conditions of DD.

 
Daily and Circadian Phase Analysis
Initial analysis of daily and circadian phase revealed rhythmic transcripts that were either accumulating or peaking during the day and those peaking during the night in the LD cycle. However, pineal transcripts rhythmically expressed in DD were clustered using a K-means clustering algorithm (GeneSpring), according to phase of mRNA abundance. The clustering analysis revealed a diverse pattern of mRNA phasing, with five clusters of peaking transcript abundance across the entire circadian day (Fig. 4Go). For each cluster a representative trace is shown indicating the average profile off all oscillating transcripts in each respective cluster. Cluster 1, which comprises 10% of the unique DD transcript set, is indicative of transcripts peaking at approximately the onset of subjective dawn circadian time (CT)-22, CT-2. Cluster 2, 26% of the unique DD transcripts, at late subjective day CT-10. Cluster 3, 50% of the unique DD transcripts, at mid subjective day CT-6. Cluster 4, 6% of the unique DD transcripts, at early subjective night CT-14. Cluster 5, 6% of the unique DD transcripts, peaked at late subjective night CT-18, CT-22. This is very similar to previous findings of other groups (27, 30). Every circadian regulated pineal gland transcript was represented in one of the above-mentioned phase clusters and is also indicated in supplemental Table 2.



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Fig. 4. Phase Cluster Analysis of Rhythmic Pineal Transcripts in DD Pineal Transcripts Rhythmically Expressed under DD Conditions Were Clustered using a K-Means Clustering Algorithm (GeneSpring), according to Phase of mRNA Abundance

The clustering analysis revealed a diverse pattern of mRNA phasing, with 5 clusters of peaking transcript abundance across the entire circadian day. For each cluster a representative trace is shown indicating the average profile off all oscillating transcripts in each respective cluster. Cluster 1 is indicative of transcripts peaking at onset of subjective dawn CT-22, CT-2, cluster 2 at late subjective day CT-10, cluster 3 at mid subjective day CT-6, cluster 4 at early subjective night CT-14, and finally cluster 5 at late subjective night CT-18, CT-22.

 
Melatonin Biosynthesis mRNAs
As has been previously shown by several authors, the mRNAs encoding several of the enzymes involved in melatonin biosynthesis are rhythmic on a daily and circadian basis. Under LD conditions, the microarray pattern indicated a low amplitude rhythm in TrH and AADC mRNA such that levels were highest in the early night. AANAT mRNA was highest during the night, whereas HIOMT mRNA peaked during the day (Fig. 5AGo). One difference was that the HIOMT rhythm amplitude was significantly greater in the microarray data than in the Northern analyses. The rhythm in TrH, AANAT and HIOMT persisted in DD with similar phase angles, whereas AADC mRNA was not significantly rhythmic in DD (Fig. 5BGo). These patterns corresponded favorably with Northern blot data employing radioactive probes derived from the microarray cDNAs corresponding to each respective gene (Fig. 5CGo). Quantification of these Northern blots, relative to ribosomal RNA (rRNA), which is not rhythmic, reveals much higher amplitude in AANAT mRNA rhythmicity than is apparent in the microarray data. However, the peak phase of AANAT mRNA levels is similar in both data sets (Fig. 5DGo).



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Fig. 5. Validation of Microarray Analysis Examining the Melatonin Biosynthesis Pathway in the Chick Pineal

A, The mRNAs encoding the melatonin biosynthesis pathway examined under LD conditions on the microarray indicated a rhythm in TrH and AADC mRNA such that transcript abundances were highest in the early night. AANAT mRNA was highest during the late night, while HIOMT mRNA peaked during the day. B, The rhythm in TrH, AANAT, and HIOMT persisted in DD with similar phase angles, while AADC mRNA was not significantly rhythmic in DD. C, These patterns corresponded favorably with Northern blot data employing radioactive probes derived from the microarray cDNAs corresponding to each respective gene. D, Quantification of Northern blots in 5C indicating the relative expression ratios of transcript vs. rRNA. The average value from two Northern blots is given.

 
Clock Genes
Several mRNAs encoding putative orthologs of genes associated with circadian clock function were identified in the cDNA libraries and represented on the microarray. These included the putative positive elements Clock, Bmal1, and Bmal2 and sequences corresponding to the putative negative elements Cry1, Cry2, and period 3 (Per3). Consistent with their putative function and consistent with published data, these mRNAs oscillated rhythmically in both LD and DD, although with overlapping phases (Fig. 6Go, A and B). These circadian rhythms and their phases were confirmed by both Northern analysis (Fig. 6Go, C and D) and ISH (data not shown). In addition, period 2 (Per2), timeless (Tim) (accession no. AY046570), and doubletime (Dbt) (accession no. AY046571), which is encoded by casein kinase I epsilon in mammals, were examined on the array. Per2 yielded an inconsistent or no signal on the array and therefore was arrhythmic in our study, although our own northern (Fig. 6Go, C and D) and published data (35) indicate per2 to be rhythmic in the chick pineal gland with a peak in mRNA amplitude at approximately CT-2–6. This was also the case for tim, which yielded an inconsistent or no signal on the array and was arrhythmic. The inconsistent or no signal results for these transcripts are likely an underestimation in the amount of PCR product spotted on the array for these transcripts, given the number of hybridizations performed in the current experiment. Dbt, however, gave a clear and consistent signal and was found to be arrhythmic in the chick pineal gland (data not shown).



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Fig. 6. Validation of Microarray Analysis Examining Chick Pineal Clock Genes

A, Orthologs of the mammalian clock gene mRNAs examined were expressed rhythmically in LD. B, This rhythm persisted in DD, although with overlapping phases. C, These circadian rhythms and their phases were confirmed by Northern blot analyses. D, Quantification of Northern blots in 6C indicating the relative expression ratios of transcript vs. rRNA. The average value from two Northern blots is given.

 
The phases of peak mRNA abundance shown here in composite are nearly identical to the peak phases of mRNA abundance of each of these genes published separately by our and other labs (17, 18, 35, 36). It is difficult to ascribe physiological roles for gene products based solely upon mRNA abundance; protein levels and activity, and physiological studies for each gene product will be necessary for a precise ascription. However, the present data are at least consistent with a putative role for these clock genes in regulation of melatonin biosynthesis (see Fig. 1Go) because putative positive elements clock and bmal1 peak at ZT/CT 10–14, coincident with the time at which AANAT mRNA should be induced, and putative negative elements cry2, per2, and per3 peak at ZT/CT 22-6, essentially in antiphase with the positive elements. In contrast, the peak values for cry1, which is expressed specifically in the pineal gland (see Fig. 9BGo), at ZT/CT 10–14 are inconsistent with a role as a negative element in this system. Obviously, more work at the cell physiological level will be required to affirm this assertion.

Retinal and Phototransduction Transcripts
Several components of the photoreceptive/phototransduction pathway were examined on the microarray, yielding many rhythmic profiles under either a LD cycle or conditions of DD (Fig. 7Go, A and B). Those examined here include pinopsin (Opsp) (16), fascin2 (Fsc2) (37), plekstrin homology domain receptor 1 (Phr1) (38), interphotoreceptor retinoid-binding protein (Irbp) (39), calretinin (Clb2) (40), early-undifferentiated retina and lens gene (Eurl), accession no. AF162861, and chick purpurin (Purp) (41). Additional potential elements of phototransduction examined but not significantly rhythmic under our criteria include transducin {gamma}-subunit (42), photoreceptor outer segment all-trans-retinol dehydrogenase (43), retinaldehyde-binding protein (44), and retinal short-chain dehydrogenase/reductase 2 (45).



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Fig. 7. Identification of Photoreceptive/Phototransduction Elements Oscillating in the Chick Pineal

A, Photoreceptive/phototransduction pathway elements shown include pinopsin (Opsp), fascin 2 (Fsc2), plekstrin homology domain receptor 1 (Phr1), interphotoreceptor retinoid-binding protein (Irbp), calretinin (Clb2), early-undifferentiated retina and lens gene (Eurl), and chick purpurin (Purp). B, These transcripts were also examined under conditions of DD.

 
Immune Function
Included among the cell types comprising the chick pineal gland (46) are B-lymphocytes, which function in immune response (47, 48). We examined several components of immune function responses and determined many to be rhythmic under a LD cycle and constant conditions (Fig. 8Go, A and B). These include tissue factor pathway inhibitor 2 (49), B cell-associated protein 2 (Bcap2) (Fig. 9CGo) (50), natural killer tumor recognition factor (51), retinoblastoma binding protein 6 (Rbbp6) (52), retinoblastoma tumor suppressor (Rbts) (53), antioxidant protein 2 (Aop2) (54), and B cell translocation gene 1 (Btg1) (accession no. NM_173999). These genes all exhibited peak mRNA expression at the LD transition (ZT-12) and remained in similar phases under conditions of DD.



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Fig. 8. Identification of Immune Function Responses Oscillating in the Chick Pineal

A, Several components of immune function responses were examined and determined all to be rhythmic under a LD cycle. These include tissue factor pathway inhibitor 2 (Tfpi2), Bcap2, natural killer tumor recognition (Nktr), retinoblastoma binding protein 6 (Rbbp6), retinoblastoma tumor suppressor (Rbts), antioxidant protein 2 (Aop2), and B cell translocation gene 1 (Btg1). B, These transcripts were also examined under conditions of DD. They all exhibited peak mRNA accumulation at the LD transition (ZT-12) and remained in similar phases under conditions of DD.

 
Protein Synthesis and Turnover
Many mRNAs encoding ribosomal proteins, proteins involved in protein synthesis and elongation and proteins involved in protein turnover, including those involved in ubiquitinylation are expressed on a daily and circadian basis. Presumably, some of these are involved in the biosynthesis of the enzymes that produce rhythmic melatonin, and others are involved in the proteosomal proteolysis of at least AANAT (55, 56, 57). It is known, for example, AANAT is synthesized on a daily and circadian schedule such that de novo transcription and translation are required each circadian cycle. Further, it is known that proteosomal proteolysis is largely responsible for AANAT degradation at the end of the subjective night and in response to acute illumination. It is likely that these processes are similarly involved in the de novo biosynthesis of transthyretin, clock gene products, photopigments, and immune function proteins.

Conservation of Rhythmic Genes across Kingdoms
It has generally been assumed that different organisms use their clocks to control species-specific events; thus, similarities within output pathways in organisms from different kingdoms would not have been predicted a priori. The recent widespread use of microarrays to profile rhythmic genes in distinct species and tissues has demonstrated the incredible diversity of clock-regulated functions. As predicted, in overall outline there is very little overlap between cycling genes in different organisms and even among tissues; however, several surprises with regards to conservation of clock-controlled genes and processes are beginning to emerge from the data. For example, comparisons of the clock-controlled transcriptome between chicks (this study) and the filamentous fungus Neurospora crassa (Ref.26 ; and Correa, A., Z. Lewis, A. Greene, I. March, R. Gomer, and D. Bell-Pedersen, manuscript submitted) reveals several genes that are regulated in both organisms (Table 1Go), including genes involved in protein synthesis, metabolism, and protein processing. These genes are conserved with remarkable sequence identity ranging from 71–96% at the nucleotide level, so that these sequences undoubtedly represent true orthologs between these diverse species. The one exception in this data set is cytochrome C oxidase, subunit 2, for which there is nonoverlapping sequence.

The observation that several ribosomal protein-encoding genes are under clock control in these as well as other organisms confirms the significance of translational regulation in clock function. This influence may occur at both the levels of output genes and central clock components. Similarly, the role of ubiquitin-mediated protein turnover in clock function is well established (58). However genes involved in metabolism and other cellular functions that are common to both organisms, such as the putative senescence associated gene, provide important clues for determining key pathways that are regulated by clocks in diverse organisms.

The emerging picture is that the avian pineal gland orchestrates a large array of rhythmic transcriptional events. These, of course, include the mRNAs involved in melatonin biosynthesis, which is the only known function of the gland. The presence of rhythmic mRNAs of chick orthologs of clock genes previously identified in Drosophila and several mammalian species raises the real possibility that these genes and their products are either directly or indirectly involved in the generation of the circadian pattern of melatonin biosynthesis. However, as stated above, more physiological research on this aspect of pineal function will be required to confirm this.

More surprising is the profoundly rhythmic expression of phototransduction sequences, protein biosynthetic sequences, and mRNAs involved in stress responses, such as the heat shock proteins, and immune function. Because it has been shown that the chick pineal gland is a lymphopoietic organ (47, 48), it is likely that the local regulation of lymphoid activity translates into both daily and seasonal regulation of lymphatic and immune activity in this species. Several studies on rodent immune function have similarly pointed to daily and seasonal changes in B- and T-lymphocyte activity (59, 60, 61). These changes have been associated with melatonin’s actions on these cells in the blood (62, 63, 64). The present data suggest that the clock directly regulates pineal lymphocytes, either through local melatonin action and/or by other paracrine signals.

In summary, the present study confirms and extends the dynamic regulation of melatonin biosynthetic and clock gene expression using a cDNA microarray technique, thereby validating our microarray as an accurate reflection of pineal activity. This microarray approach will therefore be a very useful tool in analysis of pineal clock function in the future. However, it raises in addition the possibility that the clock(s) present in the avian pineal gland are directly responsible for more rhythmic processes than has been previously appreciated. These processes may serve as springboards for further physiological research on the holistic avian and vertebrate circadian organization.


    Materials and Methods
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 Materials and Methods
 REFERENCES
 
Experimental Animals
Chicks were obtained from Hyline International (Bryan, TX) and housed under a 12-h light, 12-h dark schedule for 7 d with food and water continuously available. For cDNA library production chicks (n = 25/per time point) were killed by decapitation and their pineal glands pooled. For microarray hybridizations chicks (n = 5/timepoint/experiment) were killed by decapitation and their pineal glands pooled. Three separate pineal RNA sampling cycles were performed for array hybridizations. All DD dissections were performed using an infrared viewer.

Animals were treated in accordance with PHS guidelines; these procedures have been approved by the Texas A&M University Laboratory Animal Care Committee (AUP no. 2001-163).

cDNA Library Production
Chicks were housed in a 12-h light, 12-h dark cycle for 1 wk. On d 8 at midday (ZT-6) and midnight (ZT-18), chicks were killed, decapitated, and their pineal glands removed and pooled. mRNA was extracted using a MicroPolyA Pure Kit (Ambion). The mRNA was subsequently used to generate two cDNA libraries using a Lambda Zap cDNA Library Synthesis Kit (Stratagene, La Jolla, CA). Approximately 5000 clones from each library were selected, sequenced and organized into a database, and subjected to PCR amplification for microarray printing.

The cDNA clones were stored in 384-well plates and were propagated in four 96-well plates for plasmid production. Simplified alkaline lysis plasmid minipreps were conducted in each 96-well plate, and plasmids obtained therein were used as templates for DNA sequencing reactions. Sequencing reactions were carried out using SK primer and Big Dye Terminator Cycle Sequencing Ready Reactions (Applied Biosystems, Foster City, CA) in AB9700 96 well thermocyclers (Applied Biosystems). Reaction products were analyzed on ABI 377 automated DNA sequencers. The resulting sequence chromat files were then processed through the EST pipeline.

The EST data pipeline consisted of a Dell PowerEdge 2400 running Linux 7.3, networked to the campus local area network (LAN)/Internet and to an internal, private LAN, consisting of three shuttle cubes networked to the private LAN. The system ran the following software: 1) "Phred" read the DNA sequence trace, called bases and assigned quality values to the bases; 2) "Cross-match" created a vector-masked version of the sequence; 3) "Phrap" constructed a contig sequence as a mosaic of the highest quality reads; and 4) "BLAST" was run on vector-masked ESTs using a local copy of the NCBI protein database.

Microarray Production
The cDNA microarrays were prepared based upon approximately 4000 PCR products from each of the two pineal cDNA libraries constructed, for a total of 7,988 cDNAs present on the array. The cDNA in plasmids from each library were PCR amplified using flanking primers (SK and T7), purified by ethanol precipitation using ammonium acetate, and placed in the wells of 96 well plates at a concentration of 50 µg/ml in 3x saline sodium citrate (SSC). A GeneMachines OmniGrid microarrayer equipped with 8 Telechem SMP3 pins was used to spot the samples onto poly-L-lysine coated slides (CEL Associates, Pearland, TX). Duplicate 100-µm diameter spots were placed at intervals of 190 microns (center to center). Slides were printed in batches of 50–100. Printing was accomplished at 70 C and 60% humidity. The OmniGrid’s operating software used information on the position of the individual clones within the 96-well sample plates and the order in which the plates were used to create a file describing the position of each cDNA clone on the array for later analysis. Slides were stored desiccated at room temperature until their subsequent use. Before hybridization, the dried spots were hydrated gently over a steaming water bath, snap dried, and the DNA was UV cross-linked using a Stratalinker (Stratagene).

Microarray Hybridizations
To determine a rhythmic transcriptional profile of the pineal gland, total RNA was harvested using Trizol reagent (Gibco-BRL, Grand Island, NY) every 4 h for 1 d in a 12-h LD cycle (lights on 0600 CST; lights off 1800 CST) and for 1 d in DD. Times of sampling began 2 h after lights on (ZT-2) and continued every 4 h hence (ZT-6, ZT-10, ZT-14, ZT-18, ZT-22). When lights would have normally turned on, the timer was disabled, and birds were placed in DD. They were then collected again every 4 h, and designated CT-2, CT-6, CT-10, CT-14, CT-18, and CT-22. This sampling procedure was performed on two separate occasions. A total of four experimental microarray hybridizations were conducted for each time point, two from each respective biological sampling procedure.

The total RNA samples were then amplified to produce aRNA using a MessageAmp Kit (Ambion). Randomly primed fluorescent probes were produced from aRNA samples using Genisphere 3DNA Array 350RP expression array detection kit. The fluorescent dye on probes derived from the experimental aRNA was Cy5, whereas the dye on control probes was Cy3. In all experiments, the control sample was derived from pineal glands harvested at midnight (ZT-18) under LD conditions.

Hybridizations and washes were conducted as suggested by Genisphere. The labeled arrays were scanned in an Affymetrix 428 array scanner, and tif images were made of both Cy5 and Cy3 specific fluorescence on the array. The tif images were subsequently analyzed by GenePixPro software (Axon Instruments, Union City, CA). Information from the gal file and the .tif image were then combined to determine the Cy5 and Cy3 fluorescence of each spot as well as the corresponding background fluorescence. The program also generated a false color image of the combined fluorescence. Cy5 fluorescence is colored red, Cy3 green, and an equal mix of each is yellow.

Northern Blot Analysis
Total RNA was isolated from pineal glands using Trizol reagent (Gibco-BRL) as described by the manufacturer. Northern blots were performed as follows. Total RNA (10 µg each lane) was fractionated on 1.5% agarose/0.66 M formaldehyde gel, and probed. All probes were from labeled with [{alpha}-32P] dATP by random priming (DECA Prime II kit, Ambion) and hybridized overnight at 42 C and then washed in SSC. At least two blots were produced for each gene studied. The final wash was at 42 C in 0.1x SSC containing 0.1% SDS for 30 min. Blots were exposed to x-ray film (Biomax MS, Kodak, Rochester, NY) for 2–3 d and their images scanned and analyzed using Scion Image (Scion Corp., Frederick, MD). Data were normalized for variation in RNA loading and transfer efficiency by comparison to the 18S ribosomal band.

ISH
Animals were killed by decapitation; brains were removed and rapidly frozen in -40 C isopentane. ISH techniques were carried out as previously reported (18). Briefly, sections were fixed in 4% paraformaldehyde, followed by deproteination, and acetylation. Slides were then hybridized with sense and antisense cRNA probes for per2, cry1, bcap2, and transthyretin. Probes encoding region 512-1249 bp of the open reading frame for per2, the 3'-UTR of cry1, 293–612 bp of the open reading frame for bcap2, and 56–375 bp of the open reading frame for transthyretin were generated in the presence of [{alpha}-33P] deoxyuridine triphosphate, in vitro with Sp6 and T7 RNA polymerases for sense and antisense probes, respectively. Sections were incubated overnight at 53 C and then subsequently washed in SSC and then dehydrated in 100% ethanol. Sections were exposed to BioMax MS film (Kodak), for 48 h.

Data Analysis
The fluorescence values and background calculated by the GenePix program were saved as .gpr files. These files were then further analyzed by GeneSpring 5 (Silicon Genetics, Redwood City, CA). GeneSpring allows the data to be normalized in a variety of ways, allows the assignment of parameters and interpretations, and then allows the data to be filtered to determine differential expression. The current analysis used intensity dependent LOWESS normalization. Intensity dependent normalization is just one technique used to eliminate dye-related artifacts in two-color experiments such as this. At each time point, the results for each gene were reported as an average obtained from four microarrays. The data were reported as the normalized ratio of Cy5 (experimental) to Cy3 (control at ZT-18). Thus, the data is reported for each time point relative to the abundance of the same gene at midnight (ZT-18).

Rhythmic genes were defined as those transcripts with a statistically significant 2.0-fold or higher amplitude change in mRNA levels over a 24-h period relative to the genes’ respective mRNA abundance level at ZT-18.


    FOOTNOTES
 
This work was supported by PO1-NS39546 (to V.M.C., D.B.P., and T.L.T.), and a Texas A&M Clocks Fellowship (to M.J.B.).

Abbreviations: AADC, Aromatic amino acid decarboxylase; AANAT, arylalkylamine-N-acetyltransferase; Bcap, B-cell-associated protein 2; BLAST, basic local alignment and search tool; Cry1 and Cry2, cryptochrome 1 and 2; CT, circadian time; DD, constant darkness; EST, expressed sequence tag; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HIOMT, hydroxyindole-O-methyltransferase; 5HT, serotonin; 5HTP, 5-hydroxytryptophan; ISH, in situ hybridization; LAN, local area network; LD, light-dark; NAS, N-acetylserotonin; SSC, saline sodium citrate; ZT, Zeitgeber time.

Received for publication April 4, 2003. Accepted for publication July 14, 2003.


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 Materials and Methods
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