RTN3 Regulates The Expression Of CXCR4 And Is Required For Migration Of Primordial Germ Cells

Primordial germ cells (PGCs) are known as the gamete precursor cells in mammals, which actively migrate during embryonic development from the position where they are specified towards the site of the genital ridge where they will differentiate into male or female gametes, whose unique role is to transmit genetic information between generations. In this process, the accuracy and precision migration of PGCs determine their ultimate fate. Mis-localization of PGCs could induce the sexual dysfunction, sterility, or testicular germ cell tumors.PGCs migration process is jointly regulated by variety of factors. Previous studies on RTN3 are mostly concentrated in apoptosis and neurodegenerative diseases. We believe that identification of new molecular regulating PGCs migration is important for understanding the mechanism of PGCs movement and for developing therapies to treat diseases resulting from aberrant PGCs migration. So, we investigated the roles of RTNs in PGCs movement. In our studies, we screened CXCR4-interacting proteins using yeast two-hybrid screening to identify the proteins that interact with CXCR4 and potentially affect its functions in PGC migration. The results showed that CXCR4 can interact with RTN3 in yeast cells. To verify this interaction, a co-immunoprecipitation (co-IP) assay was performed and the interaction between CXCR4 and RTN3 detected by the yeast two-hybrid screen was confirmed. By constructing RTN3 deletion mutant, we found the RHD of RTN3 is an important domain in interacts with CXCR4.To further understand the relationship between CXCR4 and RTN3, CXCR4 expression level was detected using quantitative real-time PCR and Western blot. The results showed that the mRNA and protein expression levels of CXCR4 could be regulated by RTN3. Endogenous CXCR4 expression level of mRNA and protein could be upregulated by RTN3 overexpression or downregulated by RTN3 knockdown. Futherrnore, immunofluorescence assays show that subcellular localization of CXCR4 can be altered by RTN3. When CXCR4 and RTN3 were transfected into HeLa cells separately, CXCR4 protein mainly localized on the plasma membrane, but after co-transfect CXCR4 and RTN3 into HeLa cells, the membrane localization of CXCR4 significantly reduced and formed punctate structures in the cytoplasm, these aggregates and punctate typically co-localized with RTN3. These results indicate that RTN3 directly interacts with CXCR4 and may mediates some biological functions of CXCR4 via regulation its sub-cellular localization.To further investigate whether RTN3 can impact the biological function of CXCR4 and lead to mislocalized PGCs. we increased CXCR4b or RTN3 translation by microinjecting CXCR4b or RTN3 mRNA into 1-cell zebrafish embryos respectively, and examined the effect of this overexpression on PGC migration in zebrafish. These results showed that, embryos injected with RTN3 mRNA had PGCs probabilistic appeared in random locations outside the genital ridge, distributed throughout the body in ectopic positions. A similar phenotype was observed in the embryos injected with CXCR4b mRNA, in which PGCs lost the correct migratory pathway and were seldom found at the proper position, indicated that RTN3 overexpression can significantly affect migration in zebrafish PGCsTaken together, our results suggested that we have identified a new CXCR4-interacting proteins, RTN3, which can regulate CXCR4 expression and translocation. Moreover, increased RTN3 signaling in zebrafish can disrupt normal PGC migration. These results indicate that RTN3 may play a role in PGCs migration via interaction with CXCR4.