The pig brain proteome Regional classification based on RNA expression Background

The pig brain proteome

Within mammalian evolution, the brain has undergone changes that resulted in increased capacities to process information and perform higher cognitive functions in primates and humans. These evolutionary processes not only involve size expansion, but also variation in gene-expression. To discover human specific moleuclar features of the brain we need to first identify the shared molecular architecture of the mammalian brain. Therefore, in addition to our mouse brain efforts, we included the pig brain to include a brain that in size and structural organization is closer to the human brain than the mouse. Many of the pig proteins have high homology with human orthologues allowing us to explore and compare pig and human brain. The regional organization of brain anatomy separates the brain into regions, sub regions, nuclei and layers of specialized cells, enabling the specific function of each individual region. Transcriptomic data from the different regions facilitates classification of the regional expression variation within pig brain. Here we analyse the pig brain in a similar manner as the human and mouse brain, enabeling transcriptomic comparison between three different mammalian species.

Figure 1. Sagittal schematic drawing of the pig brain indicating the regional organzation in different colors

Regional classification based on RNA expression

The transcriptome analysis shows that 13097 of the human orthologues (n=14656) are expressed in the pig brain, and 501 of these genes show a regionally elevated expression. 5014 human genes are missing pig one-to-one orthologue and is therefore missing pig expression data in the human protein atlas, expression data based on pig genes are downloadable here.

12596 genes are classified as low regional specificity in pig brain
Only 240 are regionally enriched
Cerebellum is the region with most regionally enriched genes (n=91)
Many group enriched genes are shared among the forebrain regions, see Figure 2.

Table 1. The 10 regions of the brain and numbers of genes detected above cut of, indicating expression in that brain region, as well as number of genes classified as elevated in each region compared to the others based on transcript abundance in the individual regions (max NX of sub regions is used as representative). Same classification rules are used for the regional classification as the classification based on tissue types.

	Pig
Brain region	Detected	Regionally elevated
Cerebral cortex	12398	86
Olfactory region	12531	93
Hippocampal formation	12540	98
Amygdala	12364	51
Basal ganglia	12550	124
Thalamus	12270	28
Hypothalamus	12526	44
Midbrain	12635	93
Pons and medulla	12452	78
Cerebellum	12200	142

Table 2. The 12 genes with the highest level of regional enriched expression within the pig brain and the regional distribution category. RS-score (Regional Specificity score) corresponds to the score calculated as the fold change to the second highest region.

Gene	Description	Predicted location	RS-score
SLC6A3	Solute carrier family 6 member 3	Membrane	138
HOXB5	Homeobox B5	Intracellular	128
FRMD7	FERM domain containing 7	Intracellular	103
ADORA2A	Adenosine A2a receptor	Intracellular,Membrane	92
FAT2	FAT atypical cadherin 2	Membrane	66
HOXA4	Homeobox A4	Intracellular	60
MPZ	Myelin protein zero	Intracellular,Membrane	58
HCRT	Hypocretin neuropeptide precursor	Secreted	57
CBLN3	Cerebellin 3 precursor	Intracellular,Secreted	47
GABRA6	Gamma-aminobutyric acid type A receptor alpha6 subunit	Intracellular,Membrane	44
DBH	Dopamine beta-hydroxylase	Intracellular	38
IL16	Interleukin 16	Intracellular,Secreted	37

In order to illustrate the relation of the different brain regions, a network plot was generated, displaying the number of genes shared between different regions. The majority of group enriched genes are shared between the forebrain regions. Cerebellum and basal ganglia are the regions with largest number of regionally enriched genes. For more information and examples about the regionally elevated expression in the different regions, please visit the individual summary pages; olfactory bulb, cerebral cortex, hippocampal formation, amygdala, basal ganglia, hypothalamus, thalamus, midbrain, pons and medulla as well as cerebellum.

Figure 2. An interactive network plot of the regionally enriched and group enriched genes connected to their respective enriched region (black circles). Red nodes represent the number of regionally enriched genes and orange noders represesnt the numcer of genes that are group enriched. The sizes of the red and orange nides are related to the number of genes displayed within the node. Each node is cliacle and results in a list of all enrhcied genes connected to the hightlighted edges. The netwoek is limited to group enriched genes in combinations up to 4 regions, but the resulting lists show the compleate set of group enriched genes in the particular region

Background

The pig brain transcriptomics project is a collaborative project between human protein atlas and the Lars Bolund institute of regenerative Medicine (Dr. Yonglun Luo), BGI-Qingdao, China.

Animal information

Pig brain used for mRNA analysis were collected and handled in accordance with national guidance for large experimental animals and under permission of the local ethical committee (ethical permission numbers No.44410500000078 and BGI-IRB18135) as well as conducted in line with European directives and regulations. The experimental minipigs (Chinese Bama Minipig) were provided by the Peral Lab Animal Sci & Tech Co.,Ltd (Permit number SYXK2017-0123). Male (n = 2) and female (n = 2) Chinese Bama minipigs (1 year old), were housed in a specific pathogen-free stable facility under standard conditions. Pigs were deeply anesthetized and slaughtered by terminal bleeding. The entire pig brain was quickly removed from the scull and submerges into ice-cold PBS buffer for 2 minutes to remove excess blood and stiffen the tissue before dissection.

Transcriptomic analysis

The brain was cut in coronal slabs at the level of 1) frontal lobe/olfactory tract, 2) optic chiasm and 3) between hypothalamus and cerebral peduncle. Slaps were divided in 2 hemispheres exposing all main brain structures. For mRNA analysis, pieces of cerebral cortex and cerebellum were collected based on a sampling strategy collecting a representative sample containing all cell layers. All other regions were dissected and collected completely. Two samples (somatosensory cortex and periaqueductal gray) are missing from female 1 due to the fact that these two regions could not be identified with 100% certainty, and thus were excluded. Duplicate samples were taken from olfactory bulb from female 2, resulting in totally 119 brain samples and additional 8 samples (retina and pituitary gland), all in all 127 samples. All samples were stored at -80° C until RNA extraction within one month.