Molecular, functional, and gene expression analysis of zebrafish Ror1 receptor

Ror family of receptor tyrosine kinases ROR1 and ROR2 plays crucial roles in animal development by regulating cell proliferation, differentiation, and migration, as well as survival and death by acting as a receptor or co-receptor for Wnt5a and mediating Wnt5a-induced activation. Compared with our extensive understanding of ROR2, our knowledge of ROR1 is limited. In this study, we characterized the zebrafish ror1 gene and determined its temporal and spatial expression and biological activity. Sequence comparison and phylogenetic analyses indicate that its protein structure is similar to its mammalian orthologs. During embryogenesis, the ror1 mRNA levels were relatively low or undetectable at 6 and 9 h postfertilization. In adult fish, ror1 mRNA was most abundantly expressed in the ovary and testis. The levels of ror1 mRNA in non-reproductive system tissues were very low or barely detectable. Spatiotemporal distribution of ror1 and its ligand wnt5a in the ovary was then investigated. Reverse transcription PCR on isolated follicle layers and denuded oocytes demonstrated that both wnt5a and ror1 were exclusively expressed in the oocyte but not in the follicle layers. During oogenesis, the ror1 mRNA levels were relatively low from I to IV stage oocytes and increased dramatically at V stage oocyte. Unlike ror1, the wnt5a mRNA levels were increased gradually from I to V stage oocyte. When Ror1 was co-transfected with Wnt5a and Wnt3a in HEK293T cells, the Wnt3a-induced Wnt reporter activity was inhibited by Ror1 in a dose-dependent manner. Taken together, these results provide new information about the structural and functional conservation, spatial and temporal expression, and biological activity of Ror1 in a fish model organism.