Phenotype of homozygous mutant(Ednrbm1yzcm mice) and its small spleen phenotype
A heterozygote male mouse with abdominal white spot was mutated in EDNRB gene (c.857 T > C) which obtained by ENU mutagenesis screening. The homozygous mice (Ednrbm1yzcm mice) were obtained by genotyping and cross breeding between heterozygous mice with mutations, as shown in figure (Figure 1A). Ednrbm1yzcm mice have irregular black coats on the head and tail, and the hair color in the other parts was white.
After dissecting different genotypes mice at the age of 9 days, we found significant differences in spleen size (Figure 1B). Spleen tissue sections and H&E staining (Harris hematoxylin and eosin-y staining) were performed on mice having different genotypes(Figure 1C). Result demonstrates that the spleen microarchitecture structure of EDNRBm1yzcm mice was abnormal, compared with wild-type mice, the white and red pulps of the spleen were significantly decreasing. The white pulp was stained by blue, and the red pulp is stained by red. In the spleen of normal mice, the white pulp follicular mass and the surrounding scattered red pulp were visible, while the spleen of homozygous mice (Ednrbm1yzcm mice) lacked the white pulp follicular structure, and the loss of red pulp is more pronounced.
The genotype of Ednrbm1yzcm mutant was significantly correlated with the spleen-kidney ratio
Spleen and kidney of 5 groups of mice with different genotypes were randomly selected for weighing and statistical analysis. As shown in Table 1, the spleen and kidney weights of Ednrbm1yzcm mice (CC type) are significantly smaller than mutant heterozygous (CT type) and wild type (TT type). Therefore, EDNRB gene mutation (c.857 T > C) is related to the size of spleen and kidney in mice. The spleen-kidney ratios of Ednrbm1yzcm mice (CC) were significantly lower than those of mutant heterozygous (CT type) and wild type (TT type). SPSS analysis revealed a P-value of 0.005 that denotes the significant difference. Thus, EDNRB gene mutation (c.857 T > C) is related to the development of spleen size in mice.
Differential gene expression in EDNRBm1yzcm mice and wild-type mice
The differential genes of all the comparison groups were merged and used as a differential gene set, the different gene sets were clustered, and the genes with similar expression patterns were clustered [ Additional file 1]. The mainstream hierarchical clustering was used for clustering analysis of genes’ FPKM values, as shown in the clustering heat map( Figure 2A). HM (EDNRBm1yzcm mice) and WT (wild-type mice) groups could be seen as two different treatment groups with significant differences in gene expression.
According to the overlap of differentially expressed genes between different comparative combinations, Venn diagram was drawn to screen the differentially expressed genes common or unique to certain comparative combinations. The comparative combination treatment group and control group were expressed genes Wayne figure, as shown in Figure 2B. The Ednrbm1yzcm mutant and wild-type mice had a total of 12,359 differentially expressed genes [ Additional file 2]. However, by adopting the use of standard | log2 (FoldChange) | > 1 & Padj < 0.05 after screening, 121 differentially expressed genes, comprising of 85 upregulated genes and 36 down-regulated genes was revealed, as shown in Figure 2C.
Genes differentially expressed in mice with different genotypes are enriched in the Hippo signaling pathway that are related to the small spleen phenotype of mice
From the results of GO (Gene Ontology; http://geneontology.org/) enrichment analysis [Additional file 3 sheet 1], the most significant 30 terms were selected to draw histogram (Figure 3A). Biological processes was primarily focused on the cellular response to extracellular stimulus and amine metabolism collagen metabolic process, multicellular organic macromolecule metabolic process and other functions related to cellular response and metabolism. Cell composition was principally concentrated in pre-autophagosomal structure, lytic vacuole and lysosome, which may be due to the immune function of spleen tissues. Molecular functions are specifically concentrated on heat shock protein binding, Oxidoreductase activity, cysteine-type endopeptidase inhibitor activity involved in apoptotic process etc. It is mainly associated with enzymes redox activity, binding of various molecules and apoptosis.
KEGG(Kyoto Encyclopedia of Genes and Genomes; https://www.genome.jp/kegg/) is a comprehensive database integrating genomic, chemical and systematic functional information. With Padj less than 0.05 as the threshold of significant enrichment, the most significant 20 upregulated KEGG pathways were selected from the KEGG enrichment results[Additional file 3 sheet 2] to draw a histogram for display (Figure 3B). Upregulated genes were significantly mediated in p53 signaling pathway, Hippo signaling pathway, beta-alanine metabolism, characterization-multiple species, Arginine and proline metabolism, Amino sugar and nucleotide sugar metabolism, where Hippo signaling pathway inhibits cell growth, and upstream membrane protein receptors in the Hippo signaling pathway act as receptors for extracellular growth inhibition signals. Pan D suggested the core Hippo kinase cascade integrates multiple upstream feedbacks, that enabling dynamic regulation of tissue homeostasis in animal development and physiology[18]. Once the extracellular growth inhibition signals are sensed, a cascade of kinase phosphorylation reactions are activated and terminating the phosphorylation of downstream effectors YAP and TAZ. Cytoskeletal proteins binds to phosphorylated YAP and TAZ, holding it in the cytoplasm and reducing its nuclear activity, thus attaining the regulation of organ size and volume. Therefore, it could be associated to the reduction of spleen tissue.
The 20 most significantly down-regulated gene enrichment pathways were selected(Additional file 3 sheet 3, Figure 2A). Predominantly selected pathways are Cytokine-Cytokine receptor interaction, Osteoclast differentiation and Histidine metabolism, Renin - angiotensin system, RNA polymeras, Fatty acid elongation, Chemokine signaling pathway. Cytokine-Cytokine receptor interaction and Chemokine signaling pathway are of significant importance in guiding immune cells to and within lymphoid and non-lymphoid tissues[19]. Chemokine of Chemokine signaling pathway is a family of small molecular cytokines whose primary function is to attract immune cells to the inflammatory site[20]. This signaling pathway highlights different types of Chemokine and its receptors, as well as how Chemokine signaling activates the JAK/STAT, Ras, ERK and Akt pathways. These down-regulated genes in pathways associated with immune function may be related to the development of immune tissue structures in the spleen..