The Brain

The brain controls body movements, receives and processes sensory information, as well as higher cognitive functions. The main signaling unit of the brain are neurons, supported and protected by different types of glial cells (oligodendrocytes, microglia and astrocytes). Brain tissue includes different cell types as well as the space between the cell bodies, often referred to as neuropil, the meshwork of exons, dendrites, synapses and extracellular matrix that embed the cells of the central nervous system.

Figure 1. Schematic drawing of the pig brain showing the main brain regions in different colors.

In the pig transcriptomics analysis the brain is represented by 28 different sub regions, as well as dorsal and ventral spinal cord. In total, 30 samples from the central nervous system are used for quantitative RNA measurements. When comparing brain expression to other tissue types of the pig body, the highest expression value among the 30 sub regions is used as representation of the pig brain.The gene classification strategy highlights genes with an elevated level of expression in one or a group of tissues compared to all other tissues.

2930 genes are classified as brain elevated out of which 409 genes are highlighted as brain enriched.

Brain elevated expression

Gene expression in the brain is categorized based on two gene classification strategies, tissue distribution and tissue specificity. Figure 2 summarizes the number of genes in respective category. In total, 18358 genes are detected above cut off (1NX) in the pig brain. The tissue specificity category described 2930 genes with elevated expression in the brain compared to other tissues, and the tissue distribution category highlights 94 genes that are detected only in the brain. Table 1 shows the overlap for the brain elevated genes and tissue distribution category.

Figure 2, (A) The distribution of all genes across the five categories based on transcript specificity in brain as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in brain as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 1. Number of genes in the subdivided categories of elevated expression and tissue distribution in brain

Brain enriched expression

Genes classified as brain enriched are related to the function of the brain. The list of genes includes several known neuronal markers such as AVP, NEUROD6 and GABRA6, as well as glia specific proteins. The oligodendrocyte specific MOG shows enriched expression in the brain compared to other tissue types, and is detected in all brain regions with variable expression level. Highest observed expression level for MOG is detected in the white matter rich region corpus callosum. Neuronal proteins, such as CAMKV and CELF5 are both classified as brain enriched and detected at variable levels in the different brain regions, with low expression level in corpus callosum.

Table 2. The 10 genes with the highest level of enriched expression in brain. "mRNA (tissue)" shows the transcript level in brain as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in brain and the tissue with second highest expression level.

Immunohistochemical labeling of pig brain enriched proteins, from left to right: MOG is detected in pig cerebral cortex using HPA021873 and shows the expected location in myelin sheaths, CELF5 is detected in neuronal cell bodies, here shown in pig cerebral cortex using HPA060336 and PRKCG is detected in Purkinje cells of the pig cerebellum using HPA054560.

Expression variation within the brain

The detailed dissection of the 30 samples representing the brain enables an analysis based on the regional expression profile of the brain. Regions of the brain can be grouped into major regions and compared (as previously done in the Brain Atlas), alternatively all 30 sub regions can be compared with each other (further described here). The later analysis resulted in 4635 genes highlighted as variable within the brain sub regions, interestingly 2991 genes highlighted as within brain variable are also classified as elevated in other tissues than brain on the whole pig body comparison.

Examples of regional variation within the brain

As mentioned above, proteins related to oligodendrocytes and white matter of the brain often show higher expression in white matter rich regions, such as corpus callosum, thalamus and spinal cord, resulting in a regionally variable expression profile in the brain. Besides the already mentioned MOG, MAG and PLP1 are also examples of proteins with regionally variable expression due to having functions related to myelin that is highly abundant in the white matter of the brain. Similarly, neuronal specific markers often show low expression in the white matter rich regions, which includes only limited numbers of neuronal cell bodies. The cerebellum is a neuron dense region with several types of interneurons and a unique neuron called Purkinje cells. CA8, ARHGEF33 and PPP1R17 are all examples of Purkinje specific proteins (defined by protein detected in human brain in the Brain Atlas) and all three show cerebellum elevated expression in the pig cerebellum. Brain regional specific proteins such as the hypothalamic proteins OXT, AVP and HCRT are also examples of genes classified as brain enriched with high regional variation.

Genes classified as group enriched

Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, including brain, compared to all other tissues. There are 564 genes classified as group enriched that includes brain. In order to illustrate the relation of brain tissue to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types.

Figure 3. An interactive network plot of the brain enriched and group enriched genes connected to their respective enriched tissues (grey circles). Red nodes represent the number of brain enriched genes and orange nodes represent the number of genes that are group enriched. The sizes of the red and orange nodes are related to the number of genes displayed within the node. Each node is clickable and results in a list of all enriched genes connected to the highlighted edges. The network is limited to group enriched genes in combinations of up to 3 tissues, but the resulting lists show the complete set of group enriched genes in the particular tissue.

Examples of proteins shared between the brain and other tissues

The synaptic protein SYP shows enriched expression in brain, retina and pineal gland, tissues with neuronal function. Similarly, TH is an important protein for catecholamine biosynthesis, which has been classified as group enriched with high expression in the brain and the adrenal gland. In contrast, the nuclear SATB2 protein is important for development of cortical neurons and is also a biomarker for colorectal cancer thanks to its specific expression in large intestine. SATB2 is classified as group enriched in brain and large intestine.

Immunohistochamically labeled pig tissues, from the top, CALB2 is detected in pig brain (represented by cerebellum) and retina using HPA007305, GNG13 is detected in cerebellum (regionally specific) and retina using HPA046272 and SATB2 is detected in cerebral cortex and colon using CAB068231.