Abstract: In vertebrate embryos, gastrulation is the fundamental morphogenetic movements. It leads to the formation of the basic germ layers and the body axis. During gastrulation, convergence and extension (CE) movements narrow a group of cells mediolaterally and lengthen them to facilitate the elongation of the anteroposterior axis. In this process, the planar cell polarity (PCP) pathway, also called the noncanonical Wnt signaling pathway, is of particular importance to control CE movements. However, the precise cellular and molecular mechanisms underlying this process remains to be further studied. Rspo1 (R-spondin 1) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling levels with Wnt ligands, through which Rspo1 promotes angiogenesis and specifies hematopoietic stem cells, as well as promotes female development. Recently, it was reported that Rspo1 exhibits Wnt/β-catenin independent roles. For example, Rspo1-Lgr4-cAMP-Erα axis regulates estrogen receptor expression, and RSPO1-LGR5 activates TGFβ signaling in colon cancer. Given the complex role of Rspo1, we speculate that Rspo1 is involved in the early embryonic development. To investigate the developmental role of Rspo1 in zebrafish embryos, we examined the spatiotemporal expression pattern of rspo1 using RT-PCR and whole-mount in situ hybridization. The results showed that rspo1 mRNA is maternally deposited and expresses ubiquitously in early embryonic stages before 12hpf (hours post fertilization), implying that Rspo1 may play an important role in the regulation of embryonic development. Next, we carried out gain-of-function and loss-of function analysis of Rspo1. The results showed that either overexpression or knockdown of rspo1 abrogates the CE movements during gastrulation. In order to explore whether rspo1 affects CE movement by participating in Wnt/PCP signaling pathway, we used AP-1 luciferase reporter to monitor Wnt/PCP in zebrafish embryos. The results showed that forced expression of rspo1 decreases but knockdown of rspo1 increases Wnt/PCP signaling reporter activity, indicating that Rspo1 inhibits Wnt/PCP signaling pathway. Consistent with this result, the phosphorylated-JNK levels, an indicator of activity of Wnt/PCP signaling pathway, dramatically increased in rspo1 morphant embryos at gastrulation stage. Further analyses indicate that coinjection of rspo1 mRNA and dnJNK (Dominant negative JNK) mRNA, or coinjection of rspo1 MOs and wnt11/wnt5b mRNA synergistically enhanced CE defects. Taken together, these results suggest that Rspo1 regulates CE movements during gastrulation by negatively regulating the Wnt/PCP signaling in zebrafish embryos. Overall, our studies will provide novel insights into the regulation of Wnt/PCP signaling in vertebrates.