General characteristics of genes enriched in specific cortical regions
Recent microarray studies on mammalian brain have demonstrated very high similarities in global gene expression across different areas of the adult cortex, and in human and primate brain, the inter-individual transcriptome variation in samples from the same area tends to be much larger than variation between different cortical regions in one individual [17–20]. Still, various areas of the cortex have quite distinct functions, suggesting that certain genes are differentially expressed in each area to support its specific tasks, although few such factors have been identified so far.
We here present 65 genes that display enriched expression in the frontomedial- (FMCx), temporal- (TCx) or occipital (OCx) cortex of the adult rat brain. These highly selected genes were identified by the use of two independent microarray platforms, applying a strict statistical approach, combined with manual re-inspection of the resulting gene expression profiles and in part validation by QPCR. In support of our findings, several of the identified regionally enriched genes have previously been described to display patterned expression in developing or adult cortex, e.g. Lxn, Rorb, Nr2f1, Lmo4 and Odz3 (see below) [8, 14, 33–35].
It is important to note that the concept of cortical subregion-enriched expression does not equal cortex-specific expression; the genes identified in this study may show co-expression or even co-enrichment in other regions of the brain outside the cerebral cortex and other tissues. Thus, the genes enriched in the different cortical areas also differ in their expression patterns in other regions of the brain, illustrated here by the co-enrichment of several genes in FMCx and OCx as well as in FMCx and hippocampus. In line with this situation, ten of the regionally enriched cortical genes identified by us (e.g. Ldb2, Gpr88 and Mab21|l) were also reported to show differential expression in human and mouse motor cortex, caudate nucleus and cerebellum . Furthermore, Gpr88, a rat TCx gene showing strong co-expression in striatum (Figure 3), was identified as a human caudate- and mouse striatum-specific gene by Strand et al. .
The largest contrasts in gene expression levels were found when comparing FMCx and TCx. It is possible that the genes co-enriched in FMCx and OCx in reality could represent medially enriched genes, perhaps with a high medial - low lateral gradient-like distribution. If this is the case, then it is equally possible that TCx-enriched genes are laterally enriched rather than being specific for the TCx. In situ images from the Allen Brain Atlas suggest this situation for at least Lxn and Rorb. Gradient-like distribution of gene expression in the adult cortex has previously been discussed as an alternative to region-specific expression .
Functional roles of sub-regionally expressed genes in the cortex
The majority of the 65 sub-regionally enriched genes were involved in signal transduction processes, including cell communication, cell surface receptor mediated signal transduction and intracellular signalling cascades, but also in developmental processes, such as embryogenesis and neurogenesis (see Figure 6). This highly significant over-representation of certain biological functions suggests that the selected genes have important roles in maintaining specialised neuronal functioning in the cortical areas, supported by the fact that strikingly many of the 65 genes already have been classified and annotated. The importance of cell signalling genes in cortex has previously been reported when comparing human and primate cortical areas , and genes coding for proteins involved in signalling processes also constitute a large percentage of genes differentially expressed between other brain regions [20, 38].
In line with this, a majority of the genes were markedly enriched in neurons rather than in astrocytes and oligodendrocytes, and about two-thirds displayed laminar expression by analysing in situ images in the Allen Mouse Brain Atlas, as was observed for e.g. Fxyd6 and Lmo4 (see Figure 2). Our finding suggests that the genes may have specific functions at certain layers of the relevant cortical areas, e.g. by being expressed in only one or a few types of neurons.
This notion is further supported by our re-analysis of data from Sugino and co-workers, who have examined gene expression among 12 different populations of neurons in the adult mouse forebrain . A high proportion of the 65 genes were represented in their data set, and interestingly, about three-fourths of these showed significant differential expression across seven neocortical neuronal populations (e.g., C1ql3, Lmo4 and Odz3). Many of the represented genes were enriched in glutamatergic populations, of which eight were among the FMCx-enriched genes (e.g., C1ql3 and Lmo4). The functional specificity of an area may be conferred by certain cells or layers within that region. It is therefore plausible that many of our genes may be involved in the execution of region-specific tasks.
It is also worth noticing that according to re-analysis of data from Stead et al. , more than two-thirds of the regionally enriched genes examined show a peak or an increase in expression level in rat frontal cortex around or just after birth (e.g. Fxyd6 and Grp), as compared to less than one-third of a representative selection of house-keeping genes.
We have further mapped the 65 genes to studies comparing gene expression in different regions and at different stages of the developing mouse cerebral cortex [5, 6, 12–14], but we were unable to detect much overlap in regional enrichment (not shown). It has been indicated that "cortical" regions with unclear borders and/or immature laminar structures may not be converted to area-specific cytoarchitectures with sharp limits until the postnatal stages of development , thus regional enrichment of our 65 genes might not yet have been established at these stages.
Regionally enriched cortex genes and brain function: Selected examples
Many of the 65 genes presented here have previously been shown as involved in, or required for, normal development and function of the brain. Their preferential expression in certain parts of the cortex could be imprints of gradient like transcription patterns or related to execution of locus-specific tasks. Transgenic or mutant mouse lines have been generated for several of the regionally enriched cortex genes. Many of the knock-out mutants are lethal or have severe phenotypes in multiple tissues, especially within the brain and CNS (see Additional file 10).
Nr2f1, a well known transcription factor playing a crucial role in patterning of the neocortex, is highly expressed in the caudate part of the murine neocortex during development [8, 39], corresponding well with our finding of regional enrichment in the OCx in the rat. Cortical knock-out of the murine Nr2f1 was shown to induce massive expansion of frontal areas, including the motor cortex. These areas then occupied most of the neocortex, paralleled by a marked compression of the sensory areas to caudal OCx. These findings demonstrate that Nr2f1 is required for balancing the patterning of the neocortex into frontal/motor and sensory areas by repressing frontal/motor area identities and to specify sensory area identities . Another gene thought to be involved in neocortical arealisation and displaying high caudal to low rostral graded expression, Odz3 , was also identified to be enriched in the OCx in our study.
The genes encoding the LIM-related proteins Lmo4 and Ldb2 were both found to be enriched in the FMCx. The LIM domain is an approximately 55-residue cysteine-rich zinc-binding motif mediating protein-protein interactions. Lmo4 has two LIM domains and may play a role as a transcriptional regulator, whereas Ldb2 has a LIM-binding domain that interacts with Lmo4 . Both of them most likely function as enhancers to bring together diverse transcription factors . The two interaction partners Lmo4 and Ldb2 seem to follow each other closely in most samples analysed in the present study (Figure 2). Ldb2 appears to be more prominent in OCx than Lmo4, but both genes show a strong preference for neurons rather than oligodendrocytes and astrocytes, and both are enriched in glutamatergic neurons of the cingulate cortex (Figure 2). Interestingly, in situ hybridisations indicate an enrichment of Ldb2 in deeper layer neurons, while Lmo4 appears more prominent in layers 2/3 (Figure 2). LMO4 has previously been shown to be differentially expressed between left and right perisylvian cortex at early stages of human cortical development . This asymmetry disappears at later stages, and our data do not indicate asymmetrical expression in the adult rat cortex (Additional files 3 and 5). Lmo4-mutant mice die in utero , while in a cortical knock-out model, the boundaries of cortical functional areas were perturbed . Expression of cortical regional markers was changed and the somatosensory barrel subfield was shrinked. Thus, Lmo4 is essential for cortical development in mice, correlating well with its dramatic increase in expression level at the day of birth (Figure 2).
The FMCx-enriched gene Grp (also known as bombesin) is apparently important for the normal function of both the human and the mouse brain, and it has been shown that blockade of bombesin-like peptide receptors impair inhibitory avoidance learning in mice . In the developing rat frontal cortex, Grp expression peaks around postnatal day 7 (Figure 2). A similar increase is not observed in hippocampus or hypothalamus, suggesting that Grp might be important for late cortical development.
The TCx-enriched gene Lxn has been identified as a molecular marker for regional specification in the rat neocortex, and is produced in a subset of neurons in the lateral but not dorsal neocortex, more specifically in glutamatergic neurons in the infragranular layer . It has been suggested that the area- and lamina-specific distribution of the Lxn-expressing subpopulation of glutamatergic neurons is a distinctive feature that may contribute to the functional specialisation of the lateral cortical areas . This regional specification occurs very early in cortical development, prior to thalamocortical interactions and the completion of neurogenesis . We found that expression levels of Lxn increase dramatically around postnatal day 1 in frontal cortex, hippocampus and hypothalamus (Figure 3), despite its reported regional specificity early in cortical development. In situ images from the Allen Brain Atlas confirm the regional specificity in deeper layer neurons of the lateral cortex (Figure 3). Knowing that Lxn has a highly restricted pattern of expression in cortical neurons, it is interesting to note that it seems to be highly enriched in astrocytes compared to neurons (Figure 3).
Despite enormous differences in size and complexity between the neocortex of rodents and humans, certain brain functions are organised in the same areas throughout the line of mammalian species [47, 48]. This fact could imply that such functionally distinct areas have developed unique structures to cope with the need for different information processing and that expression of area specific genes is associated with the functional specialisation. When analysing a sub-set of the 65 regionally enriched genes in a pilot study of eight human cortical regions (dorso-lateral prefrontal cortex, primary motor cortex, primary sensory cortex, primary visual cortex, Broca's area, superior temporal gyrus, Heschl's gyrus and Wernicke's area) from three individuals, we were unable to observe the same regional enrichment as seen in adult rat (data not shown). This is in agreement with previous findings on the global level, where the variation across individuals appears far more pronounced than that observed across cortical regions in the same subject .