Connective and soft tissues

Connective and soft tissues are the tissue in between the other tissues everywhere in the body. In the pig transcriptomic analysis, adipose tissue, cartilage, synovial tissue, mesothelial tissue, vagus nerve, brain meninges and the aorta are all included and used for quantitative RNA measurements, representing different types of connective and soft tissues.

The gene classification strategy highlights genes with an elevated level of expression in one or a group of tissues compared to all other tissues. 330 genes are classified as adipose tissue elevated, 627 genes are classified as cartilage elevated, 175 genes are classified as elevated in synovial tissue, 262 genes are classified as elevated in mesothelial tissue, 275 classified as elevated in vagus nerve, 201 classified as elevated in dura mater, 571 highlighted as elevated in choroid plexus and 171 were classified as elevated in aorta compared to all other tissues.

Histological image of the pig connective and soft tissues used in the analysis can be found in the pig tissue dictionary.

Adipose tissue

Subcutaneous, abdominal and orbital adipose tissue were all sampled and analyzed. The highest expression value among the three adipose tissues was used as representation of adipose tissue when comparing to other tissue types. Gene expression in adipose tissue is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 1 summarizes the number of genes in respective category. In total, 15957 genes are detected above cut of (1NX) in pig adipose tissue. The tissue distribution category highlights 15 genes only detected in adipose tissue while 330 genes are classified as adipose tissue elevated compared to other tissues. Table 1 shows the overlap for the adipose tissue elevated genes and tissue distribution category.

Figure 1, (A) The distribution of all genes across the five categories based on transcript specificity in adipose tissue as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in adipose tissue 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 adipose tissue.

In total, there are 11 genes classified as adipose enriched, among these genes we can find pyruvate carboxylase PCthat plays a crucial role in lipogenesis and CTCFL selectively expressed by testis and adipose tissue.

Expression comparison between different adipose tissue

The expression variation category enables further details related to expression heterogeneity within the tissues that include grouped samples. In this case, expression profile from the three different adipose tissues (subcutaneous, abdominal and orbital) can be compared and highlight 382 genes with variable expression within the same type of tissue.

Cartilage

Joint (hyaline) and ear (elastic) cartilage

Gene expression in cartilage is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 2 summarizes the number of genes in respective category. In total, 15887 genes are detected above cut of (1NX) in pig cartilage. The tissue distribution category highlights 7 genes only detected in cartilage while 627 genes are classified as cartilage elevated compared to other tissues. Table 2 shows the overlap for the cartilage elevated genes and tissue distribution category.

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

Table 2, Number of genes in the subdivided categories of elevated expression and tissue distribution in cartilage.

Among the genes classified as cartilage enriched, several collagens are found, such as COL2A1 and COL11A2 as well as other cartilage related proteins, COMP, CHADL and CILP.

Table 3, The 10 genes with the highest level of enriched expression in cartilage. "mRNA (tissue)" shows the transcript level in cartilage as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in cartilage and the tissue with second highest expression level.

Expression comparison between different cartilage

The expression variation category enables further details related to expression heterogeneity within the tissues that include grouped samples. In this case, expression profile from the two types of cartilage can be compared to highlight variable expression within the same type of tissue. In total, 1362 genes show variable expression within cartilage. The elastic cartilage from the ear is rich in elastin (ELN) and elastic fiber networks, which is different from the hyaline cartilage from the joint (primarily made of type II collagen). In the expression comparison of ear (elastic) and joint (hyaline) cartilage we verified the expected high expression of ELN in ear cartilage compared to joint cartilage, as well as several less characterized genes such as MMRN2 and APOA1 with high expression in ear cartilage but not joint cartilage. On the other hand, joint cartilage showed higher expression of COL2A1, as well as TBX4 that codes for a general hyaline cartilage protein important for development, also expressed in cartilage of trachea and lung. The joint cartilage enriched expression also included several genes with brain variable expression, such as NETO2, FAM110B and KCNQ5, indicating a group of genes shared between hyaline cartilage and the brain.

Synovial tissue

The synovial tissue samples in this study was taken as close to the knee joint as possible and includes parts of the surrounding ligament, tendon and potentially some meniscus. Since the whole samples was used for RNA extraction nothing was put in fixative for further morphological examination.

Gene expression in synovial tissue is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 3 summarizes the number of genes in respective category. In total, 15143 genes are detected above cut of (1NX) in pig synovial tissue. The tissue distribution category highlights 2 genes only detected in synovial tissue while 175 genes are classified as synovial tissue elevated compared to other tissues. Table 4 shows the overlap for the synovial tissue elevated genes and tissue distribution category.

Figure 3, (A) The distribution of all genes across the five categories based on transcript specificity in synovial tissue as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in synovial tissue as well as in all other tissues. The combination of the two categories is shown in table 4.

Table 4, Number of genes in the subdivided categories of elevated expression and tissue distribution in synovial tissue.

Only 3 genes were classified as enriched in synovial tissue, and these includes RSPO4, a member of the R-spondin family of proteins with a possible function related to activation of Wnt/beta-catenin signaling pathways, G protein-coupled receptor 1 (GPR1) a possible receptor for chemerin and uncharacterized ENSSSCG00000008767.

Mesothelial tissue

Mesothelium is a simple squamous epithelium lining the body cavities, such as the pleura lining thoracic cavity and peritoneum lining the abdominal cavity. Pleura and peritoneum were sampled and used for quantitative RNA measurements, compared to all other tissues types representing the whole pig body. Gene expression in mesothelial tissue is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 4 summarizes the number of genes in respective category. In total, 15765 genes are detected above cut of (1NX) in pig mesothelial tissue. The tissue distribution category highlights 10 genes only detected in aorta while 262 genes are classified as mesothelial tissue elevated compared to other tissues. Table 5 shows the overlap for the mesothelial tissue elevated genes and tissue distribution category.

Figure 4, (A) The distribution of all genes across the five categories based on transcript specificity in mesothelial tissue as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in mesothelial tissue as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 5, Number of genes in the subdivided categories of elevated expression and tissue distribution in mesothelial tissue.

Mesothelial tissue resulted in 10 genes classified as tissue enriched, most are low abundant proteins, ALOX15 and MTNR1A are genes with higher expression level, and also show expression in adipose tissue.

Vagus nerve

Gene expression in vagus nerve is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 5 summarizes the number of genes in respective category. In total, 15028 genes are detected above cut of (1NX) in pig vagus nerve . The tissue distribution category highlights 1 genes only detected in vagus nerve while 275 genes are classified as vagus nerve elevated compared to other tissues. Table 6 shows the overlap for the vagus nerve elevated genes and tissue distribution category.

Figure 5, (A) The distribution of all genes across the five categories based on transcript specificity in vagus nerve as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in vagus nerve as well as in all other tissues. The combination of the two categories is shown in table 6.

Table 6, Number of genes in the subdivided categories of elevated expression and tissue distribution in vagus nerve.

10 genes were classified as tissue enriched in vagus nerve. Including genes with an expression overlap with spinal cord,MPZ, DHH, PLEKHA4, COL28A1 and GAS2L3. Interestingly, SHISA8 showed selective expression in cerebellum and olfactory bulb out of the dissected brain regions and PRRG3 showed a low but general expression throughout the brain regions. The remaining three vagus nerve enriched genes (NPHS1, KANK4 and GJC3) showed selective expression in the vagus nerve.

Dura mater

The dura mater is a membrane that envelops the brain, it is the outer layer of the meminges and consists of dense irregular connective tissue. The minges protects the brain and spinal cord. The dura mater includes blood vasculature and a large number of immune cells.

Gene expression in dura mater is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 6 summarizes the number of genes in respective category. In total, 15378 genes are detected above cut of (1NX) in pig dura mater. The tissue distribution category highlights 2 genes only detected in dura mater while 201 genes are classified as dura mater elevated compared to other tissues. Table 7 shows the overlap for the dura mater elevated genes and tissue distribution category.

Figure 6, (A) The distribution of all genes across the five categories based on transcript specificity in dura mater as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in dura mater as well as in all other tissues. The combination of the two categories is shown in table 7.

Table 7, Number of genes in the subdivided categories of elevated expression and tissue distribution in dura mater.

Many of the genes classified as group enriched are shared between dura mater and bone marrow, both hosting numerous immune cell types. For example EPX and NPG3, both selectively expressed by bone marrow and dura mater. In fact, many of the genes classified as dura mater enriched also showed expression in bone marrow, such as HBB, ALAS2, MMP8 in cotrast to the extracellular matrix protein

Table 8, The 10 genes with the highest level of enriched expression in dura mater. "mRNA (tissue)" shows the transcript level in dura mater as NX values. "Tissue specificity score (TS)" corresponds to the fold-change between the expression level in dura mater and the tissue with second highest expression level.

Choroid plexus

Choroid plexus can be found in all ventricles of the br, it produces and secrete most of the cerebrospinal fluid (CSF) in the brain and responsible for the active transport of substances into and out of the CSF. Fluids from the blood of capillaries is regulated by the choroid plexus ependymal cells. The choroid plexus was sampled from the hippocampal formation.

Gene expression in choroid plexus is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 7 summarizes the number of genes in respective category. In total, 15100 genes are detected above cut of (1NX) in pig choroid plexus. The tissue distribution category highlights 4 genes only detected in choroid plexus while 571 genes are classified as choroid plexus elevated compared to other tissues. Table 9 shows the overlap for the choroid plexus elevated genes and tissue distribution category.

Figure 7, (A) The distribution of all genes across the five categories based on transcript specificity in choroid plexus as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in choroid plexus as well as in all other tissues. The combination of the two categories is shown in table 9.

Table 9, Number of genes in the subdivided categories of elevated expression and tissue distribution in choroid plexus

Based on the function of chrorid

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

Aorta

Gene expression in aorta is categorized based on two gene classification strategies, tissue detection and tissue specificity. Figure 8 summarizes the number of genes in respective category. In total, 15044 genes are detected above cut of (1NX) in pig aorta. The tissue distribution category highlights 26 genes only detected in aorta while 171 genes are classified as aorta elevated compared to other tissues. Table 11 shows the overlap for the aorta elevated genes and tissue distribution category.

Figure 8, (A) The distribution of all genes across the five categories based on transcript specificity in aorta as well as in all other tissues. (B) The distribution of all genes across the six categories, based on transcript detection (NX≥1) in aorta as well as in all other tissues. The combination of the two categories is shown in table 1.

Table 11, Number of genes in the subdivided categories of elevated expression and tissue distribution in aorta

The 17 genes classified as aorta enriched all lacked gene names and only two of the pig genes holds human orthologues in this dataset. In addition, they are all low abundant proteins.

Gene expression in connective and soft tissues compared to other tissues

The overlap between the connective and soft tissues included in this study is varying. Since the mesothelial tissue includes adipocytes in the tissue composition there is an overlap in gene expression. The meninges (dura mater and choroid plexus) as well as vagus nerve differs a lot from the other tissues, and so does cartilage due to the lack of blood vessel and smooth muscle in the tissue sample of joint cartilage. In order to illustrate the relation of connective and soft tissues to other tissue types, a network plot was generated, displaying the number of genes shared between different tissue types. Group enriched genes are defined as genes showing a 4-fold higher average level of mRNA expression in a group of 2-5 tissues, compared to all other tissues.

Note, that even if both dura mater and choroid plexus are sampled from the brain they show little overlap wth brain expression profile, since they are not nervous tissue. Compared to vagus nerve that includes both neuronal axons and Scha

Figure 9. An interactive network plot of the tissue enriched and group enriched genes connected to their respective enriched tissues (grey circles). Black circles shows the connectie and softtissues representing the eye. Red nodes represent the number of tissue 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.