Comparison of regional gene expression differences in the brains of the domestic dog and human
© Henry Stewart Publications 2004
Received: 22 October 2004
Accepted: 22 October 2004
Published: 1 November 2004
Comparison of the expression profiles of 2,721 genes in the cerebellum, cortex and pituitary gland of three American Staffordshire terriers, one beagle and one fox hound revealed regional expression differences in the brain but failed to reveal marked differences among breeds, or even individual dogs. Approximately 85 per cent (42 of 49 orthologue comparisons) of the regional differences in the dog are similar to those that differentiate the analogous human brain regions. A smaller percentage of human differences were replicated in the dog, particularly in the cortex, which may generally be evolving more rapidly than other brain regions in mammals. This study lays the foundation for detailed analysis of the population structure of transcriptional variation as it relates to cognitive and neurological phenotypes in the domestic dog.
Gene expression profiling provides a novel perspective from which to consider the degree of genetic differentiation of individuals within populations. The domestic dog, Canis familiaris, is an excellent organism for this pursuit, since phenotypic and nucleotide divergence are not highly correlated. Whereas breeds are clearly, and often discretely, differentiated morphologically and behaviourally, resolution of genetic relatedness among breeds requires a large number of anonymous microsatellite markers [1–4].
The question thus arises as to whether divergence at the gene expression level is greater within or among breeds. There is a clear expectation that some fraction of the transcriptome in particular tissues and at appropriate phases of development will correlate with phenotypic variation. Similarly, disease status ought to reflect transcriptional changes,[5, 6] but any such inference must be assessed against a background knowledge of the degree of standing transcriptional variation .
Quantitative comparison of transcriptomes requires statistically orientated analytical methods that can partition the effects of multiple sources of variance. We and others have introduced linear analysis of variance algorithms for microarray data,[8, 9] and Bayesian procedures have been employed that perform similarly [10, 11]. With appropriate experimental design and moderate replication, it is straightforward to demonstrate that changes in expression smaller than twofold are significant experiment-wide. Furthermore, these approaches take into account variance contributions fromeach factor when assessing specific effects and powerfully demonstrate interaction effects. For example, in a study of the influence of sex, age and genotype on gene expression in Drosophila, we showed that a substantial fraction of the transcriptome differs more between genotypes for just one of the sexes, while age has only a very modest effect on transcription .
The objective of this study was to begin to assess the extent to which gene expression differs within and among breeds of dog in three parts of the brain. For many species, it has been shown that between 5 and 20 per cent of genes are differentially expressed between individuals, and the mammalian brain is no different [13, 14]. The left prefrontal lobe (Brodmann area 9) of three human brains differs from the homologous region of the chimpanzee brain at over 1,000 loci, although, remarkably, one human was found to differ from the others by at least as much as all three differ from three chimpanzees [15, 16]. Follow-up comparison of several regions of the human brains suggested that there is more variation among individuals than between parts of the cortex, although it is not clear whether this is due to genetic or environmental factors . Similarly, an earlier study comparing normal and postseizure mouse brains highlighted strain differences among brain regions [18, 19]. Recently, human Affymetrix chips were used to detect some divergence in transcript abundance between pools of brain tissue from several domestic dogs and two wild canid species . Here, we conducted a complementary experiment, employing a canine brain cDNA microarray to contrast region-specific expression in five individual domestic dog brains. We discuss the nature of the genes that differentiate the cortex, cerebellum and pituitary gland of C. familiaris, and argue that differences among individuals are likely to be more prevalent than are breed-specific differences.
List of differentially expressed genes by brain region.
Low in pituitary
High in pituitary
High in cerebellum
High in cortex
Low in cortex
Ribosomal protein L6
CaM kinase-like 1
Ribosomal protein L10*
Neuronal glycoprotein (×3)
Ribosomal protein L11
type III repeat
Ubiquinol-cytochrome c reductase
core protein I
Ribosomal protein L12
Dipeptidyl peptidase 7 (Dpp7)
Ribosomal protein L19
Myelin basic protein
Ribosomal protein L21
Ribosomal protein S11
Ribosomal protein S14
Ribosomal protein S25
Iodothryonine, type II
Glial fibrillary acidic
Crystallin, mu (CRYM)*
Myelin basic protein
Glucan (1, 4-alpha-)
branching enzyme 1
Aldolase C, fructose bisphosphate
Deiodinase type II
tubulin (alpha and beta)
GABA B receptor
Peanut (PNUTL2 septin)
Fas apoptotic inhibitory molecule 2
Protein phosphatase 3, beta*
Creatine kinase B subunit (×2)
Sodium-potassium ATPase, alpha*
Dissected brains from five adult dogs that were presented to the North Carolina State University Veterinary Teaching Hospital were used as the source of mRNA from pituitary gland, cortex and cerebellum. All dogs were euthanised and subjected to necropsy at the request of the owners for medical reasons. The dogs included three American Staffordshire terriers, one beagle and one American foxhound. The brains were removed in a sterile fashion within 30 minutes of death, the meninges were dissected away and tissues were taken from the frontal cortex, lateral cerebellar hemispheres and pituitary gland. These were snap frozen in liquid nitrogen and stored at -80°C. RNA isolation was performed after addition of 1 ml per 50-100 mg tissue of TRIzol reagent (Invitrogen) following the manufacturer's instructions, with further purification using an Rneasy Mini Kit (Qiagen). The quality and purity of RNA was analysed on a 0.8 per cent agarose gel and by taking 260 nm/280 nm absorbance readings on a spectrophotometer.
Raw fluorescence intensities from Scanalyze 2 were further analysed using a two-step mixed model analysis of variance procedure [8, 28] in SAS Version 5.0 (SAS Institute, Cary, NC). Raw fluorescence intensities were log transformed on the base 2 scale, and the 1,503 spots with the lowest average expression across all arrays were removed from consideration. This number was selected because they lay below the inflection point of a plot of rank-ordered average raw fluorescence intensity for all of the spots on the array. Spots at or below this point (raw values 186; log2 value 7.54) are no more intense than the mean background intensity level across all array. All of the remaining 2,721 spots were then normalised with a first analysis of variance model that adjusts for overall array and dye effects. Residuals from this model are relative fluorescence intensities (log2RFI) for each gene, essentially a measure of the fold difference in expression level for each gene relative to the sample mean for the appropriate channel on each array.
Number of differentially expressed genes by brain region.
p < 0.00002 (Bonferroni)
p < 0.0001 (FDR)
p < 0.05 (testwise)
Comparison with the Novartis Human Gene Expression Atlas  was performed using the online text query feature at http://expression.gnf.org/cgi-bin/index.cgi. This resource provides the results of duplicate (cortex and cerebellum) or single (pituitary gland) human tissue hybridisations performed against the Affymetrix Human U95A platform. Since pituitary is not represented in similar mouse data, our dog results were only compared with human. Genes listed in Table 1 that were significantly differentially regulated in the dog were individually queried. Since no statistical measures are provided online, genes whose expression was twice as high (or twice as low) in the indicated tissue relative to the other two tissues, in both species, were regarded as being consistently regulated.
For the reciprocal comparison of differentially regulated human genes, we first used the online filter to identify sets of genes in the cortex, cerebellum and pituitary that are below the average, or more than twice the average, of the 46 Novartis tissues. Pairwise comparison of these lists identifies a subset of all genes that are at least twofold differentially regulated between the tissues, which numbers between 175 and 453 genes depending on the comparison. The annotations of these genes and the canine gene accessions were then scanned for exact matches. Owing to the relatively small sample of canine genes and incomplete annotation, only around 5 per cent of the human genes could be matched to canine genes. In these cases, we asked whether the difference in expression on our arrays was significantly different in the same direction (replicated), in the same direction but not significantly so (consistent), not differentially expressed (questionable) or significant in the other direction to that seen in humans (opposite effect). Human pituitary-specific genes are apparently under-represented on our canine array, so only three clones could be compared, all of which were also upregulated in the pituitary of the dog.
Differential expression between brain regions
Of the 4,224 genes represented on our microarray, 2,721 were expressed above background levels in at least one tissue, with 591 genes showing nominal testwise significant differences (p < 0.05) in transcript abundance between the three brain regions. By contrast, at this 5 per cent significance level, just 139 genes differed among the five dogs, and 131 genes differed among the dogs in a region-specific manner, which is precisely the number of genes expected by chance. Consequently, the experiment provided good evidence for the differential expression of up to 15 per cent of the genes among brain regions but no strong evidence for differential expression between dogs.
The numbers of genes differentially expressed at significantly higher levels in one of the three brain tissues than in both of the other two are indicated in Table 2. At the significance cutoff of p < 0.0001, no significant differences are expected by chance, so the false discovery rate (FDR) is minimised. A total of 290 expression differences was observed, however: expression was elevated for 73 genes in the pituitary, 49 in the cerebellum and 22 in the cortex, while 135 genes were noticeably repressed in the pituitary and 11 genes showed their lowest expression in the cortex. Since no genes were lower in the cerebellum than in the cortex and pituitary there are thus five clusters of differentially expressed genes that appear in the two-way hierarchical cluster heat map in Figure 2. Table 2 further indicates that differential expression trends were also seen at more stringent (Bonferroni) or less stringent (testwise) significance cutoffs, confirming that gene expression was most divergent in the pituitary. The identities of the annotated genes in each class are listed in Table 1 and are discussed below.
Relative absence of differentiation among dogs
Similarly, the clustering of dogs in Figure 2 tends to indicate that any differential gene expression between dogs within brain regions also is not breed specific. Transcript abundance is remarkably uniform in the five pituitaries, while between 20 and 30 transcripts differentiate each dog from each other dog in the cerebellum. In the cortex, one of the American Staffordshire terriers is quite different from the other four dogs and there is a suggestion that the beagle and foxhound are more similar to one another than to the American Staffordshire terriers. Even though the same clustering pattern is observed when different numbers of genes are included in the analysis, it is due to just a handful of genes. Greater sampling depth and/or replication would undoubtedly elevate several percent of the genes represented in the transcriptome to the status of formally significant differential expression between individual dogs, but very few of these differences are likely to be breed specific.
Transcriptional divergence between the pituitary and the cortex and the cerebellum generally reflects the hormonal and neuronal roles of these regions of the brain. Notable among the genes with relatively low expression in the pituitary are synaptic proteins, neuronal glycoproteins and several that encode proteins and enzymes related to neurotransmitter activity. By contrast, genes upregulated in the pituitary include a thyrotropin-releasing hormone degrading enzyme, iodothyronine and multiple ribosomal proteins, consistent with the notion that the pituitary is a site of enhanced protein synthesis. Differentiation of the cortex and cerebellum is less pronounced, but includes genes with clear neuronal functions such as a glutamate transporter, fibronectin repeat protein and pentraxin, which are upregulated in the cerebellum, and an adenylyl cyclase and calmodulindependent protein kinase which are upregulated in the cortex.
Comparison of human and canine region-specific gene expression in the brain
Comparison with online data from the Novartis Gene Expression Atlas  indicates that around 85 per cent (42 out of 49) of our annotated dog genes that are orthologous to unique human genes show similar differences among brain regions. This indicates that much of the functional differentiation between the cerebellum, cortex and pituitary at the gene expression level has been retained over tens of millions of years, irrespective of differences in brain size. Among the genes highlighted with asterisks in Table 1 that do not show consistent profiles across the two species, most are members of gene families, suggesting either that precise annotation of the short dog EST sequences is misleading or that subfunctionalisation among paralogous genes occurs at a reasonable frequency .
Comparison of differential expression in human and doga
Cerebellum > pituitary
Cortex > pituitary
Cerebellum > cortex
Cortex > cerebellum
Expression variation in dogs and wild canids
As noted, no formally significant differences in gene expression between the dogs or breeds were detected. This is a little surprising, given that similar-sized studies in flies,[12, 32, 33] fish, mice  and humans [13, 15] have all found evidence for differences of approximately 10 per cent of the transcriptome between individuals. A recent comparison of pools of mRNA from three Labrador retrievers and seven German shepherds with pools from ten coyotes or five grey wolves  detected differential expression involving at least 114 genes between all three species in the amygdala and frontal lobe or between dogs and wild canids in the hypothalamus. Four of these genes were retested by quantitative reverse transcriptase polymerase chain reaction in samples from individual animals, and while two- to fourfold differentiation between species was confirmed, no differences between individual dogs were detected. Power computations indicate that detection of differential expression at levels less than 1.5-fold would, given the technical variance in our cDNA arrays, generally require more than the four replicates reported here. The trend detected in this study is that transcript abundance tends to be uniform among dogs and, as far as the very limited sample is concerned, across breeds of dogs. Nevertheless, it is likely that a broader survey encompassing different stages of brain development, or a larger sample of dogs with more replication, would detect genes whose expression varies among individuals either for genetic or environmental reasons.
It is well known from human genetics that behaviourally-related loci, such as monoamine oxidase and the serotonin transporter, are expressed at different levels among individuals [35, 36]. These genes are not represented on our array, so it is not yet clear whether expression is polymorphic in dogs as well. Close inspection of Figure 2 reveals several dozen genes whose expression is greater in two or three of the dogs than in the others and post hoc tests suggest these as candidate genes for differential regulation across individuals. Examples indicated on Figure 2 include cytochrome c oxidase subunit COX5B (our clone identity number DG1314) and a DEAD/H box protein (DG0610) in the pituitary, and a creatine kinase subunit (DG3263) and complexin (DG3512) in the cerebellum. Most of these cases show sharing of the two transcriptional states across breeds, implying that any efforts to correlate gene expression with behavioural divergence in dogs should be conducted across a broad range of breeds to avoid the effect of population stratification on inference of genetic association.
We thank Jim Mickelson for providing the sequenced cDNA clones, the owners of the dogs that provided tissues for this study and Kevin Woollard for assistance with the craniotomies. This research was supported by NIH award R01 GM61600 to GG, and by awards from the American Kennel Club Canine Health Foundation as well as the NCSU College of Veterinary Medicine to MB.
Electronic database information
The complete list of genes on our microarray, raw fluorescence intensities, as well as the results of the Mixed Model Analysis of Variance are available online at: http://statgen.ncsu.edu/ggibson/SupplInfo/SupplInfo9.htm.
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