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Physiol. Genomics 24: 163-172, 2006. First published October 25, 2005; doi:10.1152/physiolgenomics.00229.2005
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Received 9 September 2005; accepted in final form 19 October 2005.
Physiological Genomics 24:163-172 (2006)
1094-8341/05 $8.00 © 2006 American Physiological Society

Microarray screening of suppression subtractive hybridization-PCR cDNA libraries identifies novel RNAs regulated by dehydration in the rat supraoptic nucleus

Mohamed T. Ghorbel1, Greig Sharman1, Charles Hindmarch1, Kevin G. Becker2, Tanya Barrett2 and David Murphy1

1 The Molecular Neuroendocrinology Research Group, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, United Kingdom
2 Gene Expression and Genomics Unit, National Institute on Aging, National Institutes of Health, Baltimore, Maryland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The magnocellular neurons (MCNs) of the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus are the principal site of biosynthesis of prepropeptide precursor of the antidiuretic hormone vasopressin (VP). This precursor is processed during anterograde axonal transportation to terminals in the posterior pituitary gland, where biologically active VP is stored until release into the general circulation in response to physiological activation of the SON by osmotic cues. By binding to V2-type receptors located in the kidney, VP decreases the amount of water lost in urine. Osmotic activation of the SON is accompanied by a dramatic morphological and functional remodeling. We have sought to understand the mechanistic basis of this plasticity in terms of the differential expression of genes. To identify such genes, we adopted an unbiased global approach based on suppressive subtractive hybridization-polymerase chain reaction (SSH-PCR) Using this method, we generated libraries of clones putatively differentially expressed in control vs. dehydrated SON. To rapidly screen these libraries, 1,152 clones were subjected to microarray analysis, resulting in the identification of 459 differentially expressed transcripts. cDNA clones corresponding to 56 of these RNAs were sequenced, revealing many of them to be novel expressed sequence tags (ESTs). Four transcripts were shown by in situ hybridization (ISH) to be significantly up- or downregulated in the SON after dehydration. These genes may represent novel effectors or mediators of SON physiological remodeling.

osmoregulation; functional plasticity; vasopressin; expressed sequence tags


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
OSMOTIC HOMEOSTASIS IS aggressively defended in mammalian organisms. A crucial mediator of osmotic stability is the antidiuretic hormone vasopressin (VP) (31). VP is synthesized as part of a prepropeptide precursor in the cell bodies of supraoptic nucleus (SON) and paraventricular nucleus (PVN) hypothalamic magnocellular neurons (MCNs) (10), the axons of which terminate on the blood capillaries of the posterior pituitary (3). Here, mature VP peptide is stored until mobilized for secretion into the circulation by electrical activities evoked by hyperosmolality (7, 8). A rise in plasma osmolality is detected by intrinsic MCN osmoreceptor mechanisms (9, 44) and by specialized osmoreceptive neurons in the circumventricular organs that project to, and regulate, MCNs (2, 7, 8, 24, 25). On release, VP travels through the blood stream to specific receptor targets located in the kidney where it increases the permeability of the collecting ducts to water, reducing the renal excretion of water, thus promoting water conservation.

Physiological activation by dehydration is accompanied by a dramatic activity-dependent functional remodeling of the MCN, a process known as function-related plasticity (17, 38). For example, alterations in the relationship between MCNs and glia, the extent of terminal contact with the basal lamina in the neurohypophysis, the type of synaptic inputs, and the extent of electrotonic coupling between MCNs have all been documented (27, 28, 33, 36, 37, 43) and recently reviewed (33). This plasticity is governed by a complex and dynamic interplay between the intrinsic properties of the MCN, interactions between MCNs, interactions with glia, and the influences of extrinsic synaptic inputs. The response of MCNs to dehydration represents a unique and tractable model for understanding the processes whereby changes in gene expression mediate neuronal plasticity (33). However, the description of these events has, so far, been a haphazard process, dependent on the opportunistic availability of probes and the intuition of researchers (11).

Although much of the genome appears to be transcribed (14a), the number of mammalian protein-coding genes is estimated to be between 30,000 and 40,000 (41). A number of groups have started to use microarray global gene expression profiling technologies to address the question of how many of these are utilized by the MCN (29) and how the overall pattern of gene expression is changed by osmotic cues (15, 30). Note that attention has focused on the SON because it is a homogenous collection of MCNs, unlike the PVN which is a mixed tissue divided into lateral and more medial subdivisions of MCNs and parvocellular neurons, respectively. The physiological value of this approach has been illustrated by our recent identification of interleukin-6 as a novel secretory product of MCNs (15). However, a limitation of array technologies is that the analyses are restricted to known expressed sequences already archived in clone banks or databases.

To identify novel SON-expressed genes, we adopted a completely unbiased and global approach based on suppressive subtractive hybridization (SSH), first used by Bautz and Reilly in the mid-1960s (5) and subsequently improved when Duguid and Dinauer (14) showed that the ligation of generic linkers to cDNA allowed the selective PCR amplification of tester cDNA between hybridization cycles (SSH-PCR). Diatchenko et al. (13) demonstrated SSH-PCR to be effective in isolating, normalizing, and enriching differentially expressed genes over 1,000-fold in a single round of hybridization. Using this method, we generated a library of clones putatively differentially expressed in control vs. dehydrated SON. To rapidly screen this library, 1,152 of these clones were subjected to microarray analysis, resulting in the identification of 459 differentially expressed clones, 56 of which were sequenced, revealing that many were novel rat expressed sequence tags (ESTs). Four clones were selected for validation and shown by in situ hybridization (ISH) to be significantly up- or downregulated in the SON after dehydration. These genes may represent novel effectors or mediators of SON physiological remodeling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.
Adult male Sprague-Dawley rats (10–12 wk old) were maintained in standardized conditions in strict accordance with institutional and United Kingdom (UK) Home Office regulations. Dehydration involved complete fluid deprivation for 3 days (food provided ad libitum). Animal work was subject to local ethical review and was licensed by the UK Home Office.

RNA isolation.
Animals were killed by cervical dislocation, and total cellular RNA was isolated from microdissected SON using TRIzol (Invitrogen, Paisley, UK).

Generation of SON-subtracted cDNA libraries.
We generated two subtracted libraries enriched for sequences in either control (C) or dehydrated (D) SON total RNA using the SSH-PCR technique (PCR-Select cDNA Subtraction Kit; BD Biosciences Clontech, Oxford, UK). Total RNA was extracted from pools of 10–12 SON from either C or D animals using TRIzol (Invitrogen). Full-length double-stranded cDNA, synthesized using the SMART kit (BD Bioscences Clontech), was digested with Rsa I to generate short, blunt-ended fragments optimal for adapter ligation and subtraction. The cDNA in which the specific transcripts are to be found is termed the "tester," with the reference cDNA being the "driver." Aliquots of both the C and D cDNAs were then ligated to either of two different adapters (1 or 2R) to generate tester populations (C1, C2R, D1, and D2R). To enrich for differentially expressed sequences present in either C or D SON mRNA populations, two sequential hybridization reactions were performed. In the first hybridization, an excess of driver cDNA was added to each tester. After denaturation, the samples were allowed to anneal for 8 h at 68°C. Because annealing is faster for more abundant mRNA species, due to second order hybridization kinetics, remaining single-stranded tester molecules are enriched for low abundance and differentially expressed sequences. The two samples from the first hybridization were then mixed in the presence of fresh denatured driver cDNA and incubated overnight at 68°C. This further enriches for differentially expressed sequences through the generation of double-stranded tester molecules with different single-stranded adaptor sequences on each end. After dilution, hybridization mixes were subjected to PCR (Advantage PCR Kit, BD Bioscences Clontech), which first fills in the adapter ends, creating binding sites for primer annealing. Only molecules with two different primer-annealing sites are exponentially amplified. A subsequent nested PCR step further reduced background and enriched for differentially expressed sequences. PCR products were then cloned into pT-Adv Vector (Advantage PCR Cloning Kit, BD Bioscences Clontech) to produce two subtracted libraries enriched in differentially expressed cDNAs. Optimal ligation efficiency was obtained using <1-day-old PCR product with a 1:1 vector-insert ratio. The reaction was incubated overnight at 14°C. The pT-Adv vector contains a single 3' T-overhang that is promiscuous in ligating all Taq-polymerase-derived PCR products. It also contains flanking M13 forward and reverse primer sites, with the whole vector being resistant to kanamycin and ampicillin with a lacZ gene allowing simple blue/white visual identification for positive recombinant clones. Ligation mixes were transformed into TOP10F' Escherichia coli competent cells using a modified heat shock method. White colonies representing true recombinants were picked from each plate, amplified, and stored at –80°C.

Microarray analysis of the cDNA libraries generated by SSH.
The putative differentially expressed clones were arrayed on a nylon matrix to generate the SON-C-D array. cDNA inserts were amplified in a 96-well plate format using PCR. The PCR products were checked by gel electrophoresis and then transferred to 96-well "V" bottom plates, ethanol precipitated, and air-dried overnight. Pellets were resuspended in 40 µl of 1x Tris-EDTA with 0.1 M NaOH added immediately before arraying to denature the samples. We coated Nytran+ supercharge (Schleicher and Schuel BioScience, Dassel, Germany) nylon membranes with Krylon repositional adhesive (Schleicher and Schuel BioScience) and cut to the correct size, trimming the edges with a razor and wearing gloves at all times. The membrane units were placed into the arrayer, with enough to fill the entire plate. This allowed 42 microscope slide size areas to be printed. Printing of the cDNA microarray was performed using an Affymetrix 417 (Affymetrix, Santa Clara, CA) arrayer. This machine is capable of arraying 1,152 spots in duplicate, producing 42 identical arrays in 8 h of continuous printing. cDNA was spotted using a 300-µm pin and spot spacing of 665 µm center to center in 16 identical 12 x 12 grids. After spotting, membranes were individually numbered before UV cross-linking twice using a Stratalinker 2400 (BD Bioscences Clontech) at 120 mJ/cm2. As a quality control measure, arrays were inspected by sight, removed, and baked at 70°C for 1–2 h before being stored at –20°C in heat-sealed plastic bags.

Separate microarrays were probed three times using independently generated target from either C or D SON. For each replicate, SONs from 12 rats, microdissected from 1- to 2-mm coronal slices, were pooled for RNA extraction. Radiolabeled target was generated from 3–10 µg of total SON RNA (from either C or D rats) template. [{alpha}-33P]CTP was incorporated into cDNA using Superscript II RT (Invitrogen) and 10- to 20-mer poly(dT) primer (Invitrogen). Arrays were placed DNA side inward into 50-ml disposable Falcon tubes and washed with 50 ml of 2x SSC. After the draining of SSC and removal of air bubbles, membranes were prehybridized for 4 h at 50°C with 4.0 ml of Microhyb (Invitrogen) containing 10 µl of 1 mg/ml human Cot 1 DNA and 10 µl of 8 mg/ml poly(dA), both denatured at 95°C for 5 min before use. The labeled target was denatured for 5 min at 95°C and then added to the 4.0-ml prehybridization solution and allowed to hybridize to the probe at 50°C for 12–18 h. Washing of the membranes was performed in 50 ml of 2x SSC-1.0% (wt/vol) SDS at 50°C, followed by 2 x 15 min washes in 2x SSC-0.1% (wt/vol) SDS at 50°C. Moist membranes were aligned on a plastic plate, wrapped tightly in plastic, and exposed to phosphorimager screens for 1–3 days. Spot intensities were captured into ImageQuant software (Amersham Biosciences, Little Chalfont, UK) by scanning screens at 50-µm resolution on a phosphorimager (STORM, Amersham Biosciences).

NIA Neuroarray.
The NIA Neuroarray has previously been described (4, 15, 39, 40). One thousand one hundred fifty-two human cDNAs and ESTs representing various factors such as transcription factors, neuronal cell adhesion molecules, kinases, proteases, and oncogenes were spotted onto nylon membranes in duplicate. NIA Neuroarrays were interrogated in triplicate with C or D SON targets as described (see above) (15).

Microarray data analysis.
ImageQuant readings from the phosphorimager scans were first transferred to Microsoft Excel spreadsheets, predesigned to import ImageQuant information and label correct gene identities. The microarray experiment generated 2,304 volume intensities corresponding to gene expression levels obtained for 1,152 ESTs spotted in duplicate. Duplicate values were averaged for each array and then transferred into GeneSpring software version 7.0 (Agilent Technologies, Stockport, UK) for normalization and analysis. Any values below 0.01 were set to 0.01. Chip normalization was achieved by dividing each measurement by the 50th percentile of all measurements in that sample. The percentile was calculated with all raw measurements above 0.01. Gene normalization involved dividing each gene measurement by the median of its measurements in all samples. If the median of the raw values was below 0.01, then each measurement for that gene was divided by 0.01 if the numerator was above 0.01; otherwise, the measurement was thrown out. Normalized data were then subject to high-level analysis. First, data were filtered to exclude control vs. dehydration fold changes of <1.5. These data were then subjected to a Welsh t-test with a P value cutoff of 0.01.

Raw data have been submitted to the National Center for Biotechnology Information (NBCI) Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo): accession numbers GSE3132 and GSE3133.

Sequence analysis of putative differentially expressed genes.
Clones were sequenced to an accuracy of 99.5% over a single 650–700 bp read at the DNA Sequencing Facility (Univ. of Oxford, Oxford, UK) using ABI 377XL Prism DNA sequencers with ABI BigDye terminator, using universal M13 forward primer and reverse primer. With the use of a variety of computational tools, sequences were scrutinized in an attempt to determine their identity. Sequences were first compared with each other using GeneJockey software (26) to check for repetitions. Sequences were then subjected to standard nucleotide-nucleotide basic local alignment search tool (BLAST)n (http://www.ncbi.nlm.nih.gov/BLAST) (1) to determine their identity. Clones were thus classified as "known" or "novel." Novel sequences were submitted to the public database at GenBank (http://www.ncbi.nlm.nih.gov/GenBank/index.html), using the BankIt dbEST database, and accession numbers were obtained.

ISH analysis.
Brain sections were routinely probed with antisense oligonucleotides to detect transcript expression. Sense oligonucleotide probes acted as controls for nonspecific binding. However, for the novel clones identified here, the orientation of the sequence was not known. Thus probes corresponding to both strands were generated with respect to the original sequence (Table 1). Note that the two probes are not necessarily derived from the same section of the cDNA clone. By use of both probes in ISH or Northern blotting (not shown) experiments, it was possible to determine the correct coding orientation of the original sequence, as one oligonucleotide (strand 1, the anti-sense probe) would result in binding, whereas the other probe (strand 2, the sense probe) would not.


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  		Table 1. Oligonucleotide probe sequences

 
Control or dehydrated animals were decapitated; the brain was removed, frozen quickly on dry ice, and transferred to –80°C for storage until sectioning. Before the cutting, brains were removed from storage and heated to cryostat temperature (approximately –16°C) by placing them in the cryostat chamber for 1 h. Brains were mounted on Sakura Tissue Tek (Sakura, The Netherlands), and 12-µm sections were collected directly onto cold electrostatically charged mounting slides (VWR International, Poole, UK). Once three sections were placed on a slide, the slide was labeled and removed to a heating tray (37°C) for 5 min and then placed in a storage slide box. Boxes of slides were then stored at –20°C.

For radioactive ISH analysis (20), sections prepared from freshly frozen tissue were defrosted and dried on foil for ~15 min. The slides were then placed in RNase-free glass troughs, and sections were fixed with 4% (wt/vol) paraformaldehyde in 1x PBS for 5 min. The slides were rinsed twice in 1x PBS and then placed in 0.1 M triethanolamine containing 0.25% (vol/vol) acetic anhydride and 0.9% (wt/vol) NaCl for 10 min. To further permeabilize the cell membranes, the sections were dehydrated for 1 min in 70% (vol/vol), 80% (vol/vol), and then 95% (vol/vol) for 2 min and 100% (vol/vol) ethanol for an additional minute. This was followed by a delipidation wash in chloroform for 5 min and 100% (vol/vol) and 90% (vol/vol) ethanol for 1 min each. The sections were then air-dried. Forty-five microliters of the hybridization buffer containing 200,000–500,000 counts/min (cpm) of labeled probe and 1 µl of 10 mM DTT were added to each slide and covered with a glass coverslip. Oligonucleotide probes were labeled with [{alpha}-35S]dATP using terminal deoxynucleotidyl transferase (Roche Diagnostics, Lewes, UK). Hybridization buffer consisted of 50% (vol/vol) deionized formamide (Fluka; Sigma-Aldrich, Poole, UK), 0.5 mg/ml sheared salmon sperm DNA (Sigma-Aldrich), 0.4 mg/ml Ficoll, 0.4 mg/ml polyvinylpyrrolidone, 0.4 mg/ml BSA, 0.25 mg/ml yeast tRNA (Sigma-Aldrich), and 0.1 mg/ml dextran sulfate. Slides were incubated in a humidified chamber at 37°C overnight. After hybridization, the coverslips were removed by floating off in 1x SSC, and the slides were rinsed through briefly three times with 1x SSC. This was followed by three washes in 1x SSC at 55°C, 15 min each time, and two washes in 1x SSC at room temperature, 20 min each time. The sections were washed five times with 1x SSC and by a brief distilled water wash with subsequent autoradiographic visualization by exposure to Hyperfilm (Kodak, from Amersham Biosciences, Little Chalfont, UK) for 3 wk at room temperature. The autoradiographic images of the bound probes together with 14C-labeled standards (to compensate for the nonlinear response of the film to radioactivity) were measured using a computer-assisted image analysis system (Image I-22, developed by W. Rasbad, National Institutes of Health). The results were presented as integrated optical densities (area of image multiplied by average density) and expressed as arbitrary units. Sections from at least three animals, representing levels caudal to rostral through the hypothalamus, were used for each group. Analysis was performed on at least three animals and a total of >25 sections for each representative group.

For nonradioactive ISH analysis, sections were treated as for radioactive ISH, but with hybridization solution containing digoxigenin-labeled oligonucleotide. 3'-End labeling of oligonucleotides with digoxigenin was performed using a commercially available kit (Roche Diagnostics, Lewes, UK). Following standard wash protocols (see above), the slides were treated for chromogenic visualization. Sections were equilibrated in TNT buffer [0.1 M Tris·HCl, pH 7.5, 0.15 M NaCl, 0.05% (vol/vol) Tween 20] at room temperature for 10 min, and nonspecific binding was blocked by incubation with TNB buffer [0.1 M Tris·HCl, pH 7.5, 0.15 M NaCl, 0.5% (wt/vol) blocking reagent; NEN, Perkin Elmer, Beaconsfield, UK] for 30 min at room temperature. Sections were incubated with 1:100 anti-DIG-HRP (Roche) in 1x PBS for 2 h, followed by a wash, 3 x 10 min, at room temperature in TNT buffer before counterstaining in a working solution of 3,3'-diaminobenzidine (DAB; Roche Diagnostics) in peroxide buffer for 10–20 min. The reaction was stopped with a brief double-distilled H2O wash. White light images were visualized under a DMLB microscope (Leica Microsystems, Milton Keynes, UK) and captured using SPOT Basic software.

In some cases, transcripts were detected by fluorescence in combination with antigen detection by immunocytochemistry. Sections were incubated in the dark with streptavidin-fluorescein conjugate (NEN) diluted 1:100 in TNB for 1 h. The sections were then washed three times for 5 min at room temperature in TNT before further processing for co-localization using immunohistochemistry. For this, sections were washed two times in 1x PBS (10 min each time) and in 10 mg/ml BSA-0.3% (vol/vol) Triton X-100 for 30 min. The sections were then incubated in mouse monoclonal antibody PS41, which recognizes the neurophysin moiety of the VP precursor (6), at 4°C overnight. The sections were washed three times in 1x PBS, 10 min each time, after which they were incubated in a fluorescent-tagged secondary antibody for 1–2 h at room temperature in the dark. This was followed by three washes in 1x PBS, 10 min each time in the dark, after which the sections were mounted in Vectashield (Vector Laboratories, Peterborough, UK) and observed under a fluorescent DMRB microscope (Leica Microsystems), with images captured using a DC300F camera run on IM50 software (Leica Microsystems).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We used SSH-PCR to generate cDNA libraries enriched in putative differentially expressed sequences. Forward subtraction should enrich for sequences present in C tester cDNA compared with the D driver cDNA. The reverse subtraction experiment in which D cDNA acts as the tester and C cDNA as the driver ought to enrich for sequences within the dehydrated cDNA population compared with the control. The result is two subtracted cDNA populations: the forward-subtracted cDNA contains sequences specific to control cDNA, and the reverse-subtracted cDNA contains sequences that are specific to dehydration. These cDNAs were cloned to generate libraries enriched for putative differentially expressed genes. The D library should be enriched with clones corresponding to transcripts upregulated by dehydration, whereas the C library should be enriched with cDNAs derived from RNAs downregulated by dehydration. To rapidly screen out the non-differentially expressed noise, cDNA clones from the C and D libraries were arrayed to produce the SON-C-D probes. Of the 1,152 clones on the array, 512 were D clones from the D library, while 640 were C clones from the C library. SON-C-D arrays were hybridized in independent triplicate experiments to targets derived from C or D SON RNA. Data were then subject to GeneSpring analysis. Data were filtered to exclude control vs. dehydration fold changes of <1.5. These data were then subjected to a Welsh t-test with a P value cutoff of 0.01. Of the 1,152 clones on the array, 459 met these significance criteria. Two hundred sixty-two clones were putatively upregulated after dehydration. Of these, all but 23 were D clones derived from the D library, representing sequences present at a higher level in D RNA compared with C RNA (upregulated after dehydration). Of the 197 transcripts apparently downregulated after dehydration, 195 were C clones from the C library, putatively representing sequences present at a higher level in C RNA compared with D RNA (downregulated after dehydration). Thus microarray screening would suggest that the SSH-PCR libraries are highly enriched for differentially expressed genes.

To further validate our combined SSH-PCR-microarray approach to the identification of differentially expressed genes, we used GeneSpring to reanalyze data previously obtained from interrogation of the NIA Neuroarray with C and D SON targets (15). The NIA Neuroarray is an identical platform to the SON-C-D array, but, rather than representing putative SON differentially expressed genes, it bears an essentially random selection of 1,152 cDNAs corresponding to brain expressed genes. Applying the same stringent analysis to the NIA Neuroarray data revealed a single differentially expressed gene, interleukin-6 (GenBank accession no. N98591), previously identified as being upregulated in the SON after dehydration (15). This comparison would suggest that the SON-C-D array, unlike the random NIA Neuroarray, is massively enriched for differentially expressed sequences.

We then sequenced 56 clones with a putative differential expression of >2.5 fold. BLASTn analysis enabled us to identify these genes and to classify them as either known or novel (Table 2). Known sequences are defined as those already present in the GenBank database. Table 2 lists the closest homolog and, where available, the relevant Unigene identifier. Novel genes are absent from the current rat databases, although in some cases, the sequences have been identified in the rat (Rn) or, more usually, the mouse (Mm) genomes (Table 2). Note that sequence comparison revealed that some of the clones were represented a number of times in the library. This reduced the total number of genes identified to 48.


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  		Table 2. Known and novel genes

 
Because the novel clones with no significant sequence similarity to known rat gene families represent potentially interesting targets for both gene discovery and insights into the plasticity demonstrated by the SON after dehydration, we randomly selected four of them (d255, d873, d1011, and c358/c386) for further investigation and validation using ISH. Note that c358/c386 were novel when first identified but subsequently have been shown to be highly similar to mouse nuclear receptor co-activator 7 (Ncoa7).

d255 (CA865428).
Nonradioactive ISH demonstrated expression of d255 transcripts within the SON (Fig. 1, A and D) and an apparent upregulation after dehydration (Fig. 1, B and E). Radioactive ISH enabled the d255 upregulation to be quantified (Fig. 1F). A significant (P < 0.002) 2.06-fold increase in d255 RNA levels was seen within the SON. Expression is seen in the PVN (Fig. 1C), but no significant change in expression of d255 was observed after dehydration (P = 0.9201). Expression of d255 RNA is also observed at a lower level within the hippocampus, with no change after dehydration (not shown). Within the SON, expression appears to be limited to neurons (Fig. 1G).



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  		Fig. 1. Nonradioactive in situ hybridization (ISH) demonstrates expression of transcripts corresponding to novel clone d255 within the supraoptic nucleus (SON; A and D) and paraventricular nucleus (PVN; C) and apparent upregulation in the SON after dehydration (compare A and D with B and E). A and B: low magnification showing the whole hypothalamus at the level of the SON. C: expression in the dehydrated PVN. D and E: higher magnification showing the SON. Radioactive ISH enabled quantification of the upregulation of d255 transcripts in the SON (F). There is a 2.06-fold increase (a; P < 0.002) within the SON but no significant change in the PVN (b; P < 0.9201). G: high-magnification nonradioactive in situ image of the dehydrated SON. 3V, 3rd ventricle; OC, optic chiasm; C, control; D, dehydrated.

 
d873 (CA865432).
Nonradioactive ISH demonstrated expression of d873 transcripts within the SON and PVN (Fig. 2, A and C) and an apparent upregulation in both structures after dehydration (Fig. 2, B and D). Expression appears to be limited to neurons. Radioactive ISH enabled the d873 upregulation to be quantified (Fig. 1E). Significant 3.8- and 3.1-fold (P < 0.001 and P < 0.002) increases in d873 RNA levels were seen in the SON and PVN, respectively.



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  		Fig. 2. Nonradioactive ISH demonstrates expression of transcripts corresponding to novel clone d873 within the control SON (A and C) and PVN (A) and an apparent upregulation in both structures after dehydration (B and D). A and B: low magnification showing the whole hypothalamus at the level of the SON. C and D: high magnification showing the SON. Radioactive ISH enabled quantification of the upregulation of d873 transcripts in the SON and PVN (E). There is a 3.8-fold increase (a; P < 0.001) within the SON and a 3.1-fold increase (b; P < 0.002) in the PVN for this clone.

 
d1011 (CA865430).
Nonradioactive ISH demonstrated expression of d1011 transcripts throughout the hypothalamus, particularly after dehydration (compare Fig. 3A, control, with Fig. 3B, dehydrated). Quantitative ISH analyses showed that, within the region of the SON and the PVN, this upregulation is 1.8- and 1.5-fold (P < 0.003 and P < 0.04), respectively (Fig. 3C). Interestingly, expression of d1011 transcripts appears to be limited to cells with an architecture reminiscent of blood vessel. Within the SON, these blood vessel-like cells are distinct from the VP neurons (Fig. 3D).



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  		Fig. 3. Nonradioactive ISH demonstrates a robust expression of transcripts corresponding to novel clone d1011 over the entire hypothalamus after dehydration (compare A, control, with B, dehydrated). Quantitative ISH analyses showed that, within the region of the SON and the PVN, this upregulation is 1.8- and 1.5-fold (P < 0.003 and P < 0.04), respectively (C). Expression of D1011 appears to be confined to cells with an architecture reminiscent of blood vessels. Within the SON (D), these cells, illuminated by fluorescent ISH (green), are distinct from the vasopressin (VP) neurons detected by immunocytochemistry (red).

 
c358/c386 (CA865416); Ncoa7.
ISH demonstrated expression of d358/c386 transcripts within the SON and PVN (Fig. 4A) and an apparent downregulation in the SON after dehydration (Fig. 4, B and C). Radioactive ISH enabled the c358/c386 downregulation to be quantified (Fig. 4D). A significant (P < 0.001) decrease by fourfold of the mRNA for this transcript is observed in the SON after dehydration. Even though a reduction in the abundance of c386 transcripts was also observed within the PVN, this value was not significant due to a large variability in expression levels in control animals.



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  		Fig. 4. Radioactive ISH demonstrates expression of transcripts corresponding to clone c358/386 in the euhydrated SON and PVN (A). Nonradioactive ISH demonstrates expression of transcripts within the control SON (B) and an apparent downregulation after dehydration (C). Radioactive ISH enabled quantification of the downregulation of c358/c386 transcripts in the SON and PVN (D). There is a 4.0-fold decrease (a; P < 0.001) within the SON with a smaller 1.54-fold decrease within the PVN, although this value is not significant (b; P = 0.2420).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The SON is a highly specialized brain region that produces vast amounts of a peptide hormone, VP, that is destined to be released into the blood stream in response to appropriate physiological cues. The well-established responses of these neurons to a stimulus such as dehydration include major changes in their phenotypic identity, characterized by altered morphology, cell-cell interactions, synaptic inputs, electrical activity, and biosynthetic activity (33). We hypothesize that altering steady-state mRNA levels is an effective strategy for regulating and mediating these processes. To substantiate this notion, we have used an SSH-PCR-based differential hybridization strategy, combined with microarray screening, to isolate and identify novel genes that are both up- and downregulated in the SON as a consequence of chronic dehydration.

SSH-PCR is a time-consuming and complicated technique that allows the researcher to compare two populations of mRNA. Libraries of cDNA clones are obtained that correspond to transcripts that are enriched in one population compared with another. Ji et al. (19) examined the practical and theoretical limitations of SSH-PCR. Their mathematical calculations revealed that effective enrichment of a target RNA by SSH-PCR is determined by its concentration ratio between tester and driver, rather than absolute difference, and its abundance in the total mRNA population. They concluded that SSH-PCR cannot exclude all non-differentially expressed genes from a library, and that low-abundance transcripts, such as those that encode transcription factors, cytokines, and receptors, would be difficult to detect.

Although excluded from their theoretical modeling, it was noted that, because PCR amplification is also sequence dependent and nonlinear, this might also result in the over- or underrepresentation of certain sequences in libraries derived from SSH-PCR. Ji et al. (19) pointed out that

In profiling gene expression differences in diseased vs. normal tissues or over an experimental time course where small changes in gene expression are more likely to be physiologically relevant, SSH PCR would be highly ineffective in profiling gene expression changes. In such situations, differential screening of very large SSH PCR libraries can potentially compensate but at high costs in time and labour.

To overcome the limitations of SSH-PCR, a number of groups recognized the potential of using microarray platforms to rapidly screen for differentially expressed genes in extensive SSH-PCR libraries (42). For example, genes regulated by estrogen in the hypothalamus have been thus identified (22, 23). Similarly, to better to understand the differences between the phenotype of the control and dehydrated SON at the molecular level, we used microarray screening of cDNA libraries derived from SSH-PCR. This unbiased gene expression analysis generated a bank of 1,152 genes putatively enriched in one population compared with the other. Microarray analysis enabled the facile screening of this library, with the identification of 459 putative differentially expressed genes. Fifty-six clones with a differential expression of >2.5-fold were then sequenced and hence identified by comparison with the public databases. Excluding repetitions, a total of 48 genes were identified. These were classified as known genes, already present in the GenBank database, or novel genes, new to science. Fourteen of the 48 identified genes (29%) fell into the novel category. We then confirmed the differential expression of four expressed sequences using ISH.

Three lines of evidence suggest that the combined SSH-PCR/microarray method is an effective and valid approach to the identification of differentially expressed genes. First, of the 1,152 clones on the array, 459 met the significance criteria. Two hundred sixty-two of these clones were putatively upregulated after dehydration. Of these, 239 were D clones derived from the reverse subtraction library, representing sequences putatively present at a higher level in D RNA compared with C RNA (upregulated after dehydration). The remaining 23 clones were false positives derived from the C library. Of the 197 transcripts apparently downregulated after dehydration, 195 were from the forward subtraction, putatively representing sequences present at a higher level in C RNA compared with D RNA (downregulated after dehydration). Only two clones were false positives from the D library. Thus microarray screening would suggest that the SSH-PCR libraries are highly enriched for differentially expressed genes, and that the overall false discovery rate is only 25 of 459 (~5%). Second, we compared the SON-C-D microarray analysis with the NIA Neuroarray, an array constructed using an identical platform but with a random selection of gene sequences. In contrast to the 459 genes identifies with the SON-C-D array, only a single gene, interleukin-6, was identified by the application of the same GeneSpring protocol to the NIA Neuroarray. This comparison would suggest that the SON-C-D array, unlike the random NIA Neuroarray, is massively enriched for differentially expressed sequences. Finally, four putative differentially expressed genes were confirmed, using ISH, as being significantly regulated in the SON in response to dehydration.

The combined SSH-PCR/microarray methodology described here has numerous advantages over the use of these techniques alone. First, arraying of clones enables the rapid screening of the SSH-PCR libraries, with the elimination of many false positives based on stringent statistical tests. Second, by use of SSH-PCR to enrich for differentially expressed genes before arraying, a stringent process of selectivity was already started, resulting in the identification of many putative differentially regulated genes. Finally, we note that many (29%) of the transcribed sequences identified in this study are novel. These genes would never have been identified using standard microarray platforms, which are dependent on a prior knowledge, either in the form of expressed sequences archived in clone banks or sequence information in databases.

Table 2 lists the candidate genes identified in this study as being differentially expressed in the SON as a consequence of dehydration. The functions of these genes within the SON remain unknown. We note that some of these genes (Hspa8, astrotactin 1, and GABA-A receptor gamma-2 subunit) have previously been identified as being at least twofold enriched in the SON compared with whole hypothalamus (29). Cytochrome c, shown here to be downregulated in the SON as a consequence of dehydration, was previously identified by differential screening of cDNA libraries generated from supraoptic nucleus from hyper- or hyponatremic rats (16). Other known dehydration-regulated genes were not identified but may well be represented within the remaining unsequenced clones.

We decided to focus our studies of four randomly selected sequences corresponding to novel genes. However, as these studies progressed, new information in the databases revealed that one clone, c358/c386, was highly similar to mouse nuclear receptor co-activator 7 (Ncoa7) and is likely to be the rat equivalent of this gene. Ncoa7 (also known as ERAP140) (32) was isolated in a screen for estrogen receptor (ER){alpha}-interacting proteins using the ER{alpha} ligand-binding domain as a probe. The expression of Ncoa7 is cell and tissue type specific and is most abundant in the brain, where its expression is restricted to neurons (32). In addition to interacting with ER{alpha}, Ncoa7 also binds to ERß, thyroid hormone receptor (TR)ß, peroxisome proliferator-activated receptor (PPAR){gamma}, and retinoic acid receptor (RAR){alpha}. RAR{alpha} and ERß are known to be expressed in the SON. Indeed, ERß expression levels correlate inversely with dehydration (34, 35). ERß inhibits VP secretion, and its downregulation during fluid deprivation may contribute to stimulated VP release. Because Ncoa7 can enhance the transcriptional activities of nuclear receptors with which it interacts (32), its coordinate downregulation with ERß, confirmed in our ISH studies (Fig. 4), might be a component of these physiologically important regulatory pathways.

Of the 48 ESTs identified in this study, we have used ISH to confirm that 4 of them (3 of which are novel) are indeed differentially regulated in the SON after dehydration. Interestingly, whereas three (d255, d873, and c358/c386) clones showed ISH hybridization patterns suggestive of expression in neuronal elements, one, d1011, showed labeling within what appears to be the vasculature of the entire hypothalamus. Because the starting material for our SSH-PCR analysis was total RNA derived from whole SON samples, then contributions from glia and blood vessels, as well as neurons, are to be expected. The expression of D1011 within blood vessels emphasizes the point that the response of the SON, indeed the entire hypothalamus, to a physiological stimulus such as dehydration is not confined to neurons but rather involves a complex cellular network. The exact roles of these novel genes within this network remain to be determined.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Wellcome Trust (UK), the Biotechnology and Biological Sciences Research Council (UK), and the British Heart Foundation (UK). G. Sharman was a recipient of a PhD Student Fellowship from the Medical Research Council (UK).


   ACKNOWLEDGMENTS
 
Present addresses: M. T. Ghorbel, Bristol Heart Institute, Univ. of Bristol, Bristol Royal Infirmary, Marlborough St., Bristol, BS2 8HW, UK; T. Barrett, Gene Expression Group, Information Engineering Branch, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: D. Murphy, The Molecular Neuroendocrinology Research Group, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, Univ. of Bristol, Dorothy Hodgkin Bldg., Whitson St., Bristol BS1 3NY, UK (e-mail: d.murphy{at}bristol.ac.uk)

10.1152/physiolgenomics.00229.2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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