Morphology And Gene Expression Analysis Of Different Tissues From Nile Tilapia (Oreochromis Niloticus)

Nile tilapia(Oreochromis niloticus)is a cichlid tropical fish,native to Africa,is a high quality aquaculture species.Tilapia is mainly cultivated in Guangxi,Hainan and other high temperature areas after being introduced into China.It is one of the largest cultured varieties in the world,and the research is mainly focused on breeding control and diseases,but systematic histological studies are still lacking.In this paper,three different staining methods(HE PAS and Masson staining)were sampled to display the fine structure of 16 tissues,and then transcriptome sequencing methods were combined to link some tissues with gene expression.Finally,rassf4 gene was taken as an example to explore the molecular mechanism of gene selective expression.Relevant data provide important data for tilapia basic research.In this paper,the heart,liver,spleen,gills,pseudogills,intestine,esophagus,stomach,testis,ovary,eyes,brain,kidney,peritoneum,muscle and other organs of tilapia were selected for morphological analysis.The digestive tract of tilapia starts from the mouth,passes through the esophagus,stomach,intestine and ends at the excretory orifice.The structure of the esophagus from the outside to the inside is the outer membrane,muscular layer,submucosa and mucous layer.The stomach and intestines are also similar in structure to the esophagus.The top of the epithelial cells of the gastric mucosa stained with PAS was deep and purplish red,indicating that the gastric mucosa was rich in mucus cells.In Masson staining,the gastric submucosa is green,because the collagen fiber in the gastric submucosa is developed and belongs to loose connective tissue.The histological stratification of the foregut,midgut and hindgut does not differ greatly.But the anterior villi are sparse.The villi of middle intestine were longer,which increased the contact area with food.The villi of the posterior intestine become extremely short.The intestinal lamina propria is thin and the submucosa is rich in blood cells.Acid mucus was secreted in the esophagus,intestine and stomach of tilapia by PAS staining of digestive tract.Mucus not only protects the mucosal epithelium from physical or chemical damage caused by interactions with digestive tract contents or enzymes,but also plays a role in mediating biological reactions.It also contains immunoglobulins that fight bacteria in ingested substances.Gill is the main respiratory organ of fish,and gill filaments and cartilage support the gill.Mucous cells are a prominent feature of gill epithelium.The mucus-rich gill surface is conducive to the function of ion regulation,physical protection and immune protection.Tilapia have pseudogills that are covered with connective tissue,filled with blood,and have a glandular structure.Pseudogill is structurally similar to gill,but the filaments are joined together and the cartilage lies between them.In addition to excretion,kidney of tilapia also has hematopoietic,immune,endocrine and other functions.After HE staining,the wall of proximal convoluted tubule was dark red,while the wall of distal convoluted tubule was light red.The difference in staining results also proved that they had different epithelial cell structure and function.The spleen of tilapia contains red pulp and white pulp,but the boundary is not obvious,and contains a large number of red blood cells and lymphocytes respectively.In our experiment,we selected ovaries that have not yet entered the spawning stage,and oocytes at different stages of development can be seen.Spermatogonia,spermatocyte and spermatozoa at different developmental stages can be clearly seen in the spermatolobule of the testis.On the basis of transcriptome data,weighted gene co-expression network analysis(WGCNA)was performed on the genes expressed in 15 tissues of tilapia,and gene co-expression network was constructed based on the similarity of gene expression trends.Through this network,we can not only search for highly correlated gene modules,but also intuitively understand the possible expression regulation relationships among genes.Through the co-expression cluster heat map,10co-expression modules were selected,which were represented by different colors.The number of genes in these modules ranged from 101 to 1365.High expression or specific expression genes were found in each color module for analysis.For example,RAS-association domain family gene in blue module is specifically expressed in ovary and may be involved in signal transduction regulation and growth regulation.The rassf4 transcript is 1400 bp in length and consists of 921 bp open reading frame encoding 306 amino acids.The specific expression of this gene in ovary was confirmed by PCR and immunohistochemistry.Immunohistochemical results showed that rassf4 was expressed in follicular cells.The methylation rates of rassf4 promoter region(-734/-1012)in ovary and testis were 13.28% and 92.85%,respectively.Deletion of the fragment at-734 to-1012 reduced rassf4 promoter activity to 62.33%.The results showed that the methylation level of rassf4 gene promoter region regulated its differential expression in testis and ovaries.The diversity of animal tissues is the result of selective gene expression,which may be regulated by a variety of mechanisms,including DNA methylation.In this paper,morphology and molecular biology are closely combined to provide basic data for studying the molecular mechanism of tissue differentiation and function of tilapia.