Study On Function Of Wnt9 Gene During Metamorphosis In Apostichopus Japonicus

Apostichopus japonicus is one of the important species of marine cultured echinoderms in China,which has great economic value.Metamorphosis is the key stage of individual development of A.japonicus,which involves significant morphological characteristics and the transformation of body axis system.However,at present,little is known about the mechanism of head tail body axis formation and symmetry transformation in the early development of A.japonicus.Many studies has confirmed that Wnt pathway signal plays an important role in the correct establishment of body axis,growth and differentiation of Drosophilid and other model animals.This paper aims to explore the regulatory role of Wnt9 gene in the formation of body axis in the early development of A.japonicus,using Race and real-time PCR and whole in situ hybridization technologies,to study how Wnt signal and its related pathway genes β-catenin in embryonic and adult tissues of A.japonicus carries out spatiotemporal expression and spatial distribution,giving the results as follow:(1)The Wnt9 gene sequence of A.japonicus was cloned by Race,with a total length of 1603 bp,an open reading frame of 1104 bp,encoding 367 amino acids.The structure of Aj Wnt9 protein was predicted by smart software.It was found that there was a transmembrane region and a Wnt1 conserved domain.Phylogenetic tree analysis showed that the A.japonicus and the Holothuria glaberrima were grouped together,Strongylocentrotus purpuratus and Acanthaster planci were grouped together.(2)Real time PCR technology detected that Wnt9 could be expressed in multiple larval stages and tissues.In the development stage,the expression of Wnt9 in 16 cell stage was slightly higher than that in other stages from fertilized egg to gastrula.During the period from late-stage auricularia to juvenile,the expression of mRNA increased step by step,and increased significantly to doliolaria.The expression of Wnt9 in juvenile reached the highest and was several times higher than that in other stages by eleven times;The mRNA expression in the body wall in adult tissues was significantly higher than that in other tissues.The expression in muscles,intestines,respiratory trees and coelomocytes was also relatively high,but only in gonads.(3)The different expression of Wnt9 and β-catenin gene: Wnt9 was highly expressed only in the body wall of the juvenile and doliolaria;β-catenin was expressed in larval development stage,but the expression was not high,and the expression was high in respiratory tree;The expression Wnt9 and β-catenin was not synchronous in the larval stage and tissues of A.japonicus;(4)The results of whole in situ hybridization showed that the positive signal of Wnt9 gene gradually increased from late-stage auricularia to juvenile and doliolaria,and the positive signal appeared in late-stage auricularia,but there was no obvious concentration;During the juvenile stage,the positive signal was enhanced and concentrated in five pairs of globules;The positive signal of the doliolaria stage is strong and concentrated around the anterior body cavity.This showed that the expression intensity of Wnt9 increased significantly with metamorphosis,and the expression site was also significantly concentrated in the precursor cavity of doliolaria.The expression trend of β-catenin gene in three larval stages of A.japonicus was different from that of Wnt9 gene.There was almost undetectable at the late-stage auricularia,When metamorphosed to juvenile,trace expression intensity was detected and accumulated in the precursor cavity;However,no changes of β-catenin positive signal aggregation area were observed in the doliolaria.The results of this study showed that the spatiotemporal expression and spatial distribution of Wnt9 gene of A.japonicus increased significantly with the process of metamorphosis from late-stage auricularia,and the downstream gene of Wnt classical pathway β-catenin has been in a low-level continuous expression state,which indicates that Wnt9 gene plays an important role in the larval metamorphosis of A.japonicus.It may regulate cell proliferation and differentiation through non classical Wnt pathway and participate in the formation of five radiation symmetrical bodies of A.japonicus.These results provide a theoretical basis and research direction for the in-depth study of the growth,development and institutional changes of Wnt signaling pathway in the metamorphosis period of A.japonicus.