The Mutation of Ednrb(c.857 T > C) Associated With Atrophied Spleen Phenotype in Mice

Background:The endothelin signaling pathway plays an important function in the migration, proliferation, and differentiation of neural crest cells. Endothelin receptor B (EDNRB) was reported to have a small spleen phenotype in its decient mouse model .In our study, we also found that the mutation of EDNRB gene (c. 857 T > c) led to an atrophied spleen phenotype in mice. Different genotypes of EDNRB were signicantly correlated with the spleen-kidney ratio, and the spleen phenotypes of Ednrb m1yzcm mice were smaller. The results of the tissue section and H&E staining showed that the spleen microstructure of Ednrb m1yzcm mice was abnormal. In order to explore the molecular mechanism, three groups of Ednrb m1yzcm and wild-type mice were used as control, and standard |log2(FoldChange)|>1 and Padj<0.05 were used to study the inuence of EDNRB gene mutation on spleen transcriptional group in mice. GO and KEGG enrichment analysis was conducted to explore the signal pathway related to small spleen phenotype. Results: Through sequencing of mouse spleen transcriptome, 121 differentially expressed genes were selected. Results of the KEGG pathway enrichment analysis showed that in Ednrb m1yzcm mice, upregulated genes were signicantly enriched in the Hippo signaling pathway, this pathway inhibits cell growth and modulates organs size and volume; and down-regulation of immune functionally associated pathways such as cytokine receptor interaction and chemokine signaling pathway. In addition, chemokine of Chemokine signaling pathway may also be related to the development of spleen immune tissue structure. Conclusions: In the we found mice gene features such an to explore the we performed RNA sequencing we found that upregulated genes signicantly enriched Hippo

selected. Results of the KEGG pathway enrichment analysis showed that in Ednrb m1yzcm mice, upregulated genes were signi cantly enriched in the Hippo signaling pathway, this pathway inhibits cell growth and modulates organs size and volume; and down-regulation of immune functionally associated pathways such as cytokine receptor interaction and chemokine signaling pathway. In addition, chemokine of Chemokine signaling pathway may also be related to the development of spleen immune tissue structure.
Conclusions: In the experiment, we found that mice with mutations in the EDNRB gene have features such as an atrophied spleen and changes in the structure of the spleen. In order to explore the reasons, we performed RNA sequencing on three groups of Edrnrb m1yzcm and wild-type mice, and we found that upregulated genes were signi cantly enriched in the Hippo signaling pathway. And this signal Pathway is a proven signaling pathway that controls organ size and immune function, so we speculate that the size of the spleen may be related to the Hippo signaling pathway. This study provides a theoretical study of the mechanism of spleen development.

Background
The growth of the spleen begins in the fth week of development, and it originates from the mesoderm of the embryonic viscera until lymphatic colonization. The spleen acts as the hematopoietic organ [1] turning out to be the largest peripheral lymphatic organ in the body. When its organizational development completes, its structure and function becomes very complex. The essence of the spleen is distributed into three portions: white pulp, red pulp and marginal zone [2][3][4]. The white pulp is composed of dense lymphocytes, which is the primary site for the body's speci c immunity [5]. When the antigen invades the spleen and mediates the humoral immune response, a number of lymph nodes in the white pulp increases. The red pulp of the spleen is mainly composed of blood sinus and splenic cord. The slow rate of blood ow in the red pulp facilitates the complete antigens and phagocytes interaction. Marginal zone (MZ) is located at the junction of red and white medulla. The lymphocytes in this area are sparse compared with white medulla, mainly B cells, but there are more macrophages. It is an essential site for antigen capture, antigen recognition and immune response induction in the spleen [6]. Immune function is the most crucial function of the spleen. The interactions between various immune cells and immune factors in the spleen restrict each other. It can complete the body's non-speci c immune functions through phagocytosis, and T cells and B cells mediated the cellular and humoral immunity to exert speci c immune functions.
The mouse EDNRB gene is located on the negative strand of chromosome 14 and contains 29025bp [7,8]. It plays an important role in the pathogenesis of congenital megacolon, melanoma and other related diseases. This gene encodes a G protein-coupled receptor and is localized to the cell membrane under physiological conditions. As a G protein-coupled receptor superfamily [9], it binds to endothelin (ET) and transmits information into the cell through the second messenger system of intracellular Ca2 + phospholipid-dependent protein kinase to promote endoplasmic reticulum and sarcoplasma [10,11].
There is a quick surge of Ca2 + in the reticulum calcium reservoir, and Ca2 + concentration in the cytoplasm rapidly increases [12]. During embryonic development, the EDNRB / EDN3 / ECE1 signaling pathway plays an important role in the migration and development of neural crest cells into ganglion cells, affecting the formation of the colorectal and enteric nervous system [13][14][15][16]. Previous experiments have also shown that the gene affects the size of the spleen [17]. This experiment will explore the changes in the spleen phenotype after mutation of the EDNRB gene (c.857 T > C). In this experiment, a male mouse with abdominal leukoplakia whose T -C mutation at base 857 of the coding region of the EDNRB gene was obtained by ENU mutagenesis screening, and the offspring were obtained by cross-breeding with ordinary female mice, and their tail DNA was collected. As a template, homozygous mice (Ednrb m1yzcm mice) were genotyped by PCR and sequencing technology. Transcriptome sequencing was performed on homozygous mice and wild-type mice to explore their differentially expressed genes and enrichment pathways, to obtain theoretical evidence of the effect of this gene mutation on spleen size in mice.

Results
Phenotype of homozygous mutant(Ednrb m1yzcm 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 (Ednrb m1yzcm mice) were obtained by genotyping and cross breeding between heterozygous mice with mutations, as shown in gure ( Figure   1A). Ednrb m1yzcm 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 signi cant 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 EDNRB m1yzcm mice was abnormal, compared with wild-type mice, the white and red pulps of the spleen were signi cantly 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 (Ednrb m1yzcm mice) lacked the white pulp follicular structure, and the loss of red pulp is more pronounced.
The genotype of Ednrb m1yzcm mutant was signi cantly 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 Ednrb m1yzcm mice (CC type) are signi cantly 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 Ednrb m1yzcm mice (CC) were signi cantly 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 signi cant difference. Thus, EDNRB gene mutation (c.857 T > C) is related to the development of spleen size in mice.
Differential gene expression in EDNRB m1yzcm 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 le 1]. The mainstream hierarchical clustering was used for clustering analysis of genes' FPKM values, as shown in the clustering heat map( Figure 2A). HM (EDNRB m1yzcm mice) and WT (wildtype mice) groups could be seen as two different treatment groups with signi cant 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 gure, as shown in Figure 2B. The Ednrb m1yzcm mutant and wild-type mice had a total of 12,359 differentially expressed genes [ Additional le 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 le 3 sheet 1], the most signi cant 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 speci cally 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 signi cant enrichment, the most signi cant 20 upregulated KEGG pathways were selected from the KEGG enrichment results[Additional le 3 sheet 2] to draw a histogram for display ( Figure 3B). Upregulated genes were signi cantly 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 signi cantly down-regulated gene enrichment pathways were selected(Additional le 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 signi cant importance in guiding immune cells to and within lymphoid and nonlymphoid tissues [19]. Chemokine of Chemokine signaling pathway is a family of small molecular cytokines whose primary function is to attract immune cells to the in ammatory 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..

Discussion
The mice used in this study were mutant mice with EDNRB gene, and EDNRB was one of the primary susceptible genes for congenital megacolon. Moreover, subsequent anatomical experiments all con rmed that homozygous mutant mice with Ednrb m1yzcm had congenital megacolon in addition to changes in hair color and reduced spleen. Zhi Cheng reported that mice with genetic de ciency of the endothelin receptor type B displays not just colonic aganglionosis, but also manifest a profoundly abnormal immunophenotype characterized by markedly small spleens, B and T cell splenic lymphopenia, and disordered splenic microarchitecture [21]. This nding is consistent with our experimental results, which shows that the loss of function of EDNRB gene can affect the development of spleen as well as the congenital megacolon. Dang et al. reported that the AGH-Ednrb sl/sl rat model of HSCR displayed lymphoid depletion with signi cantly reduced spleen weight and total splenic cell count of less than 15% in wildtype AGH rats, with severe B, CD4 and CD8 T cells reduction [17]. In conclusion, we have shown that the Ednrb-null mutation not only presents as colonic inactivation similar to Hirschsprung's disease, but also presents as a immune abnormality characterized by signi cant reduction in spleen volume.
Similar to the T cell homing to the tumor's endothelial barrier [22], Frykman noted that the spleen was smaller in Ednrb -/bone marrow transplant recipients, suggesting that EDNRB may play a role in lymphocyte transport between bone marrow, thymus, and spleen [23]. However, the lymphocytic recirculation pathway through the spleen remains a mystery, and the lymphocytic migration pathway to the spleen is still unde ned [2]. Gosain A found that Ednrb NCC-/showed reduced spleen size and structural changes, decreased red medulla in myeloid center and marginal areas, and decreased B lymphocytes, especially IgM + IgD hi (mature)B lymphocytes, which are usually activated after antigen presentation and produce IgA [24]. The spleen is the primary source of IgA producing mature B lymphocytes; therefore the spleen may be the primary anatomic site of B-lymphocyte defects in Ednrb NCC-/-.
KEGG analysis results showed that the down-regulated genes were signi cantly enriched in the Hippo signaling pathway. Originally discovered in drosophila, the Hippo signaling pathway is considered to be a conserved signaling pathway that controls organ size during development by inhibiting cell growth and proliferation and promoting apoptosis. The Hippo pathway acts in repressing the transcription of target genes promoting proliferation and survival. And more recently, there is growing evidence that hippo signals play a crucial role in regulating the immune system. To date, various knockout mice of major components of the Hippo pathway have been elucidated the role of this pathway in the immune system. Hippo signaling pathway has been extensively studied in lymphocytes, especially T cells, and emerging results show that MST1/2 (MST1/2 kinases, the mammalian orthologs of Drosophila Hippo.) is critical for T-lymphocyte development, migration, homing, and differentiation. MST1 also regulates adhesion and tra cking of B-cell; the number of peripheral B cells, as well as splenic marginal zone B cells, was signi cantly reduced in MST1-de cient mice [25,26]. All of these suggest that the major reason for the shrinkage of the spleen is probably the effect of this signaling pathway.

Conclusions
In this study, RNA sequencing was performed on three groups of Ednrb m1yzcm and wild-type mice. The methods of KEGG pathway enrichment analysis and GO enrichment analysis enriched differentially expressed genes in different signaling pathways. We found that upregulated genes were signi cantly enriched in Hippo signaling. This signaling pathway is a proven signaling pathway that controls organ size and immune function. Therefore, we speculate that the size of the spleen may be related to the Hippo signaling pathway. Down-regulated genes are mainly enriched in Cytokine-Cytokine receptor interaction, Chemokine signaling pathway, etc. These signal pathways related to the immune function of the spleen, and the spleen is mainly composed of lymphocytes and other cells, so while the immune function is affected, it also has a certain impact on the size of the spleen. This study provides a theoretical study for the development of the spleen.

Animal
Male adult mice (SPF grade, 7-9 weeks old) with EDNRB mutation induced by ENU were purchased from College of Veterinary medicine Yangzhou University. All the mice were caged in a controlled environment. All procedures were approved by the Committee for the Ethics on Animal Care and Experiments in Northwest A&F University. To minimize suffering during sacri ce, mice were euthanized by cervical dislocation. A total of 269 mice were bred until the follow-up test, of which 23 were used in the speci c experiment: Five individuals of CC type (homozygous mutant Ednrbm1yzcm mice), CT type and TT type(wild type mice, control group) were randomly selected to analyze the correlation between genotype and spleen-kidney ratio; Two individuals of CC type and TT type(control group) were randomly selected for tissue sections and HE staining; Three individuals of CC type and TT type(control group) were randomly selected for RNA sequencing. The remaining 246 were euthanized because the genotype did not meet the breeding and testing requirements.

Acquisition of Ednrb m1yzcm mice
Three male mouse with abdominal white spot mutation were obtained by ENU mutagenesis screening , we mated them with normal female mice (N=20). female mice(wild type) in estrus are placed into the cages with male adult mice with EDNRB mutation (two females in each cage with one male), the next morning, check the female for vaginal plugs to make sure the mating is successful. phenotyping data from both sexes is collected at regular intervals on age-matched wild-type mice of equivalent genetic backgrounds (This part of the work was done by F.W). The animals were randomly assigned to mate after being genotyped, using a computer-based random sequence generator. The heterozygous male(CT N=21) and female(CT N=32) offspring mice with EDNRB gene mutation were cultured to estrus and mated to obtain the second generation of offspring mice. The mouse tail tissue was collected. The mice tail tissue DNA was extracted using the improved phenol-chloroform method[27], PCR technology was used to amplify the gene fragment containing the mutation site (the 857th base T to C of the EDNRB gene coding region), and genotyping was performed by sequencing technology to select heterozygotes containing mutations. The heterozygous male and female offspring mice with EDNRB gene mutation were cultured to estrus and mated to obtain the second generation of offspring mice. To minimize suffering during sacri ce, mice without mutations were euthanized by cervical dislocation. The mice fasted for 18 hours before autopsy, but were provided with normal drinking water, The mice were euthanized at the operating table. The mutant heterozygous mice were selected for hybridization, and the Ednrb m1yzcm mice with irregular black spots on head and hip and mice with white body were obtained. The size and sequence of the primers, and Tm (°C) are shown in Table 2.
Anatomy and organ weighing of Ednrb m1yzcm mice and wild type mice We weighed Ednrb m1yzcm mice and wild-type mice that were genotyped by sequencing at 9th day of birth, with an electronic balance, dissected their spleen and kidneys, weighed and recorded the data(This part of the work was done by M.L). Three spleens of Ednrb m1yzcm mice and three spleens of wild-type mice were rapidly stored in the refrigerator at -80℃ (for subsequent transcriptome sequencing). One one spleen from both Ednrb m1yzcm mice and wild-type mice were placed in the tissue xation solution (for the preparation of subsequent tissue sections).
Correlation analysis of genotype of Ednrb m1yzcm mutant mice and spleen-kidney ratio After 9 days of birth, the second-generation mice tail tissue was collected and their DNA was extracted as a template, and the gene fragment containing the mutation site (base 857 T to C in EDNRB gene coding region) was ampli ed by PCR. And then we genotyped by sequencing technology. Five individuals of CC type (homozygous mutant Ednrb m1yzcm mice), CT type and TT type were randomly selected. SPSS 19.0 software (Statistical Product and Service Solutions, Version 19.0 Edition, IBM, Armonk, NY, USA) for oneway analysis of variance (ANOVA) was used to analyze the signi cant relationship between different genotypes and ratio of their spleen weight to kidney weight and to know whether the base T to C mutation is related to the spleen-kidney ratio of mice(This part of the work was done by F.W.W).
Production of tissue sections.
DNA was collected from the second-generation mice tail tissue on 9th day after birth, and the gene fragment containing the mutation site (base 857 T to C in EDNRB gene coding region) was ampli ed by PCR technology. An individual of CC type (Ednrb m1yzcm homozygotic mutant) and an individual of TT type were selected, their spleen tissues were dissected and collected, soaked in tissue xation solution, para n sections were prepared, H&E (Harris hematoxylin and eosin-y) staining was conducted, and tissue section graphs were photographed with 20× and 40× resolution using CaseViewer software.

Construction and sequencing of spleen tissue
The spleens of 3 Ednrb m1yzcm mutant homozygous mice (HM1, HM2, HM3) and 3 normal wild-type mice (WT1, WT2, WT3) were sent to Novogene Co., Ltd. Standard extraction method was used to extract RNA from spleen tissues. NanoPhotometer spectrophotometer was used to detect RNA purity (OD260/280 and OD260/230 ratios) and Agilent 2100 bioanalyzer was used to accurately detect RNA integrity. After taking strict quality control measures according to the two quality control standards, library construction, quality inspection and computer sequencing were conducted.
Collation and analysis of sequencing data of spleen tissue Differential gene expression analysis Differential gene analysis was performed according to the screening method of differentially expressed genes of Novogene Co., Ltd. The standard was |log2(FoldChange)| > 1 & Padj< 0.05, and the differentially expressed multiple was located at 2 times or more, with padj value set below 0.05.

GO function and KEGG pathway enrichment analysis
ClusterPro ler software was used to analyze the GO function enrichment and KEGG pathway enrichment of differential gene sets. The target gene set (differential gene set) is mapped to the background gene set. The enrichment analysis is based on the principle of hypergeometric distribution, in which the differential gene set is a differential gene obtained by signi cant difference analysis and marked to the GO and KEGG database gene set. Background gene sets are all sets of genes that were analyzed for signi cant differences and marked to GO or KEGG databases. Padj < 0.05 was used as the threshold for signi cant enrichment.

Consent for publication
Not applicable.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and Additional les.