Transcriptome Analysis Of Wnt16 Knockout Zebrafish Model For The Study Of Skeletal Development

Skeletal development is regulated by a variety of transcription factors and signaling pathways.When the key genes for skeletal development are mutated,it may cause body deformities or bone diseases such as osteoporosis,and in severe cases,it can lead to early embryonic death.Zebrafish is a common research model,its wnt16 gene has a high sequence similarity to the human WNT16 gene.As a typical Wnt ligand,wnt16 is mainly involved in the regulation of vertebrate skeletal development and homeostasis.Therefore,this study is based on the wnt16knockout strain established in the previous research of our research laboratory,and aims to explore the effect of wnt16 knockout on zebrafish skeletal development and related signaling pathways.The main results and conclusions obtained in the study are summarized as follows:1.Genotype and phenotype analysis of wnt16 zebrafish:wnt16zebrafish was identified as a homozygous mutant by genotype,and quantitative real-time PCR(q RT-PCR)analysis found that the expression of wnt16^-/-m RNA was significantly reduced.The protein structure prediction of the mutant gene wnt16 indicated that its tertiary structure changed compared with the wild type(WT).Subsequently,whole-body in situ hybridization(WISH)was used to observe the expression of wnt16gene in zebrafish,which was mainly selectively expressed in bone tissues such as head,pectoral fin buds and pharyngeal arch cartilage.Safranin O-fast green staining results showed that wnt16^-/-cartilage was damaged,cells arranged unevenly and cell number decreased.These results indicated that the wnt16 gene was successfully knocked out and expressed in bone-related parts,which in turn affected the cartilage development and bone formation of zebrafish.2.Analysis of differentially expressed genes in the transcriptome:Transcriptome data screening yielded a total of 773 co-expressed differential genes.Gene ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis showed that down-regulated genes were mainly enriched in signaling pathways such as m TOR,Fox O and VEGF related to skeletal development,while up-regulated genes were not enriched in signaling pathways related to bone.The protein-protein interaction(PPI)network established a network relationship between wnt16 and down-regulated genes.q RT-PCR verified that the transcriptome data was reliable.The analysis results showed that knocking out the zebrafish wnt16 gene may inhibit m TOR,Fox O and VEGF signaling pathways,resulting in abnormal zebrafish skeletal development.3.Weighted gene co-expression network analysis(WGCNA):Divided the gene expression level,that is,genes with FPKM value greater than 5 into 12 modules,and correlate them with phenotypic data to obtain the yellow module with the highest correlation coefficient.Cytohubba method was used to obtain the hub genes of this module,the analysis results showed that ten hub genes such as polr1c,ska1,dsn1 and so on could be used as potential genes that affect skeletal development.This study used the zebrafish model to confirm that knockout of wnt16 gene affected skeletal development and the expression of related signaling pathways,and also discovered some potential genes related to skeletal development,which provides an important reference for the study of wnt16 gene.