2.3 Results
2.3.4 Patterns of differential expression
close to zero; there was no general trend of increase or decrease. The distribution is similar to that seen in the forebrain.The increased expression of MT3 in the hip- pocampus is particularly notable in light of the increases in MT1 and MT2 expression in the forebrain. Having identified DE genes, we next characterized commonalities among them.
Figure 2.10: Metallothionein (MT) 3 is upregulated in the hippocampus of densin knockout relative to wild-type mice. The hippocampus displays a similar pattern of DE genes as in the forebrain, although the variability is generally greater in the hippocampus. MT3 expression is increased in the hippocampus of densin mice relative to wild-type mice. In the forebrain, MT1 and MT2 are among the most highly DE genes, and like MT3 in the hippocampus, MT1 and MT2 are increased in the forebrain in densin knockout mice relative to the wild-type mice. Key: Mt3 (metallothionein 3), Igh-1b (immunoglobulin heavy chain 1b (serum IgG2c)), Rps3a (ribosomal protein S3A), Cryab (crystallin, alpha B), Hddc3 (HD domain containing 3).
DE genes. We reasoned that if expression of the DE genes was concentrated in a particular region, this might indicate a locus of change in the densin knockout mouse.
The ABA contains the results of large-scalein situ hybridization studies of thou- sands of transcripts (Lein et al., 2007). They also quantify the intensity and density of expression in different brain structures (Allen Institute for Brain Science, 2006).
Because the information was not available in the form required for our analysis, we generated scripts that parsed the XML-formatted data and aggregated it into a for- mat that could be queried. Consequently, we could compare relative expression in different brain structures for 3,225 genes.
In the densin knockout mice, 418 transcripts were DE in the forebrain with a z-score of more than two. Of these, we have ABA data for 283 of them. For each, we compared the reported in situ hybridization intensity in five mutually exclusive structures: hippocampus, striatum, thalamus, hypothalamus and midbrain. These comparisons are represented in Figure 2.11. Each row represents the intensity of one transcript in the five structures. The region with the lowest expression is represented by a thin red bar; the one with the highest with a green bar, and other structures by intermediate colors. The rows are then sorted to group common expression patterns together. As indicated by the prevalence of green in the hippocampal column, genes affected by the loss of densin are more highly expressed in the hippocampus than in the other structures analyzed.
We quantified the difference in expression between brain structures. The ABA intensity values reported for the hippocampus are significantly different from those
reported in any of the other structures; the greatest p value of t tests between values reported for the hippocampus and any other structure is less than 0.01. The same tests performed on density values indicate a trend, but not a significant difference, in density of expression; the greatest p value in those comparisons is less than 0.07. The analysis of the intensity values indicates that the genes we identified as DE in the forebrain are significantly more expressed in the hippocampus than in the hypothala- mus, thalamus, striatum or midbrain. Repeating the analysis with randomly chosen groups of expressed genes produces the same result, reflecting the high frequency of DE gene expression in the hippocampus. This suggests that the hippocampus is a promising region to look for the effects of the loss of densin.
Networks
Having identified the hippocampus as a structure of high abundance of DE genes in that structure, we sought to identify interactions between the DE genes. We queried a curated interaction network (Ingenuity Pathway Analysis) and identified networks of highly interconnected genes in that structure (Figure 2.12 on page 64). The net- work is centered around Iκb which is significantly upregulated in both forebrain and hippocampus of densin knockout mice. Networks are ranked by the fraction of DE genes involved in the network compared to the fraction expected by chance as well as by the magnitude of the difference in transcription level. Although Iκb is the most highly ranked network, it includes few of the most DE genes.
-1.5 -1.0 -0.5 0.0 0.5 1.0 1.5
1
Hipp Str Thal Hypo Mid
Figure 2.11: Genes differentially expressed in the forebrains of densin knockout mice are highly expressed in the wild-type hippocampus. We used data from the ABA to identify structures enriched for genes identified as DE in the forebrain of densin knockout versus wild-type mice. One brain region is represented in each column (Hipp=hippocampus, Str=striatum, thal=Thalamus, Hypo=hypothalamus, Mid=midbrain). Each row corresponds to a DE gene (z>2). The ABA intensity values are separately color coded for each gene. Higher expression is represented by green, and lower expression by red. The trend of bright green in the hippocampus indicates that many of the genes are more highly expressed there than in other structures. The intensity values measured in the hippocampus differ from all other structures (p<0.01). No other columns significantly differed.
Figure 2.12: The NFκB pathway is a central hub connecting many genes differen- tially expressed in the densin knockout mouse. Networks among genes DE in the forebrain of the densin knockout versus wild-type mice were identified by querying a curated interaction network (Ingenuity Pathway Analysis). The shapes of the genes correspond to the function of the genes. Green shapes indicate upregulated genes;
red, downregulated. Brighter colors indicate a greater difference in transcription level between densin knockout and wild-type mice. White shapes are genes included to demonstrate possible indirect connections. For example, the inclusion of NFκB allows a connection between ALDH7A1 and GOS2. The network shown here is centered around Iκb, which is DE in both forebrain and hippocampus of densin knockout mice. This network includes many genes implicated in neurological and cardiovascular disease.