Molecular Cloning, Expression Pattern, And Function Of R-spondin2 During Early Muscle Development In Tongue Sole Cynoglossus Semilaevis

R-spondin2 is a member of R-spondin protein family.There are four members, including Rspol-Rspo4, and all of them are secreted proteins. Several structurally distinct domains are recognized:(i) a hydrophilic, putativesignal peptide sequence for secretion, (ii) two or more cysteine-rich furin-like(CR) domains, (iii) a thrombospondin type I repeat (TSR/TSP-1) domain, and(iv) a basic amino acid-rich (BR) domain of variable length.Studies in mouse Xenopus and some other species show that Rspos can activate and indicate with wnt signaling pathway, thus it can involve the development of many important tissues,such as bone,muscle,vessel, and it can also involve the development and differentiation of limbs and gonads, maybe it can even play important roes in the formation of many diseases.For example,in the study of Xenopus,Rspo2 can activate typical Wnt pathway,and promote β-catenin-mediated muscle development,and the deprivation of Rspo2 can block this process,indicating that Rspo2 is necessary for wnt-mediated muscle development.In mouse,knockdown of key gene Nobox in oocyte formation can reduce the expression level in female gonad of Rspo2 gene,showing potential importance of Rspo2 gene in the development of ovary.Additionally,Tongue sole (Cynoglossus semilaevis) is an economically important marine fish in China,like many other flatfishes, it exhibits sexual growth dimorphism, and body weight between females and males can have significant differences.In addition,muscle development is very important for commercial fishes. So that, in our study, biological functions involved in the above two aspects of Rspo2 was explored in this fish species.In this study, we cloned the complete protein coding sequence ORF of Tongue sole Rspo2 gene and predicted the structure of this protein.Sequence analysis show it has the same domains as expected, and 3D models have shown its high identity with other species,such as zebrafish and medeka.Real-time PCR observation illustrates its low expression level until gastrulation during early embryonic developmental stages,and high levels in brain,gill and gonads for tissue expression.Topographical distribution of Tongue sole Rspo2 transcripts had an extensive distribution in gonads, which might indicate its potential function involved in gonad development. In addition, we take model organism zebrafish as a research platform, that might provide the possibility to study gene functions of marine economic fish.In this study,based on microinjection technique, combined with the zebrafish whole embryo staining and whole mount in situ hybridization experiment, we preliminarily inquiry that R-spondin2 gene can have potentially important influence for early muscle development in half smooth tongue sole.