Studies On The Scale Development And Expression Of Key Regulatory Genes In Blunt Snout Bream(Megalobrama Amblycephala)

Skin appendages refer to structures derived from the epidermis or dermis and play important roles in the survival of vertebrates.Skin appendages are not only the developmental biological models with biomedical relevance,but also have been concerned due to the evolutionary significance.Therefore,the study of vertebrate skin appendages has an important role in zoological research.As one of the fish skin appendages,scales play important roles on fish body.The molecular mechanism has important theoretical significance for explaining the development and formation of skin appendages.Fish scales developmental patterns,histological processes and molecular regulatory mechanisms have not been studied.M.amblycephala is an herbivorous economic fish species in China.In this study,we used M.amblycephala as a fish model to carry out some relted researches to reveal the biological process of fish scale development,which will provide a theoretical basis for the further study.The main results are as follows:1. In order to reveal the scale coverage pattern,the histological changes during the scale developmental process and the scale tissue structure of M.amblycephala,this study combined the morphological and histological methods,with the used alizarin red staining,calcein staining,and electron microscopy techniques to systematically observed the scale development of the M.amblycephala.The results showed that the scales of the M.amblycephala belong to the sycloid scales of the ganoid scales.The ultrastructure observation showed that the structure was divided into the limiting layer,the external elasmo and the internal elasmodine.The starting position of scales development is located at the lateral line of the trailing edge of operculum.With the continuous growth of the fish body,the scales appeared at the side of the caudal shank and the base of the pectoral fin.From the beginning of the appearance of scales to the complete coverage,its development has experienced five stages.Scales were derived from the continuous differentiation of cells between the epidermis and dermis of fish skin.The histological process mainly included:early morphogenesis,late morphogenesis,early differentiation,late differentiation and folding stage.The growth of M.amblycephala has an important influence on the development of scales,and the correlation between the development of scales and the body length of fish is higher than the day-old.2. In order to further screen the regulatory genes related to the development of M.amblycephala scales,this study analyzed the transcriptome of the skin and scale tissue at the beginning of the scales in the two stages before and after the coverage of M.amblycephala.Through transcriptome analysis,9802 differentially expressed genes were screened.The GO and KEGG functional enrichment analysis were performed on these genes.The most enriched GO items were:cellular process,biological regulation,metabolic process;there were 5452 differentially expressed genes enriched in KEGG pathway,it is divided into six branches?cell process,environmental information processing,genetic information processing,human diseases,metabolism and organic systems?,among which the signal transduction category under the category of environmental information processing The largest number of genes?1632?.86 genes related to the scales development of M.amblycephala were screened,mainly involving in integrin family members?ITGA3,ITGA10,ITGA5,ITGA4,ITGB4,ITGB6,ITGBX,ITGB3,ITGAV?,FGF signaling family?FGF20,FGF12,FGF16,FGFR1a?and mitogen-activated protein kinase family?MAP4K2,MAP4K3,MAP3K2,MAP3K13,MAP2K7,MAP3K12,MAP3K8?.3. To further explore the role of candidate genes in the scale's development of M.amblycephala,the expression patterns of candidate genes?EDA,EDAR,SHHa,BMP2b,FGFR1a,SHHb,FGFR1b?in adult fish tissues were analyzed.The results revealed that EDA,SHHb,EDAR and FGFR1b were significantly expressed in the skin tissues of M.megalobrama.The evolutionary analysis showed that the EDA gene of M.amblycephala and zebrafish were closely homologous.The zebrafish EDA CRISPR/Cas9 system was successfully constructed using gene editing technology.In the F₀ generation,the sequencing results showed a double peak appeared near the target site,indicating that the knockout was effective,which laid the foundation for the next screening of mutants.Compared with WT,the mutant individuals showed curvature in the spinal spine and pulse spine of the tail.As the fish body grows further,the ribs and spinal spine also showed some deformation in the trunk vertebrae.The scales also appeared partially missing,laying the foundation for the next screening of mutants.