| The Wnt signaling pathway is a major embryonic pathway that controls cell proliferation, cell fate, and morphogenesis in vertebrate embryos. Dysregulation of Wnt signaling pathway leads to a wide range of pathologies including cancers in human. The Wnt ligands activate two major intracellular pathways:known as the β-Catenin-dependent canonical Wnt pathway (also Wnt/β-catenin pathway) and β-Catenin-independent non-canonical Wnt pathways, such as Wnt/PCP pathway. Soon after egg fertilization, Wnt pathway components play a role in myriad biological processes, including body-axis determination and gastrulation movement.RSPOs/Rspos are a family of secreted proteins, which were discovered as canonical Wnt activator. More and more researches in mammalian and amphibian reveal the significant biological functions of RSPOs/Rspos, while the underlying molecular mechanisms remain controversial due to the conflict results. In addition, little is known about the family members, gene structure, and functions of Rspos of teleost fishes. In this study, using zebrafish as model animal we investigated in the functional study of zebrafish rspol and rspo3 genes during embryogenesis.The study about rspo3 shows that zebrafish Rspo3 has a unique domain structure, which is different from that of homologs in mammal and amphibian. It contains a third furin-like (FU3) domain. This FU3 is present in other ray-fined fish species but not in cartilaginous fish such as elephant shark. Zebrafish rspo3 is maternal deposited and has a ubiquitous expression pattern in early embryonic stages. After 12 hpf it expressed in a tissue-specific manner. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increase ventral-posterior development and stimulates ventral and posterior marker gene expression. According to the expression levels of maternal Wnt target genes, forced expression of rspo3 has no effect on maternal Wnt signaling, while knockdown of rspo3 intend to increase maternal Wnt activity. Overexpression of rspo3 abolishes Wnt3a action and reduces the endogenous Wnt signaling activity, and knockdown rspo3 results in increased Wnt/β-catenin activity. The experiment about the neural ectoderm development shows that forced expression of rspo3 promotes the anterior neural ectoderm and decreases the posterior one, while knockdown rspo3 has the opposite effects. The results above confirm zebrafish Rspo3 regulates dorsoventral and anteroposterior patterning by antagonizing the Wnt/β-catenin signaling pathway during the early embryo development. We further explore the mechanism of zebrafish Rspo3. Based on the data from genetic interaction with different canonical Wnt activators, we found that zebrafish Rspo3 negatively regulated canonical Wnt pathway functions downstream of Lrp6 receptor, upstream of β-Catenin-destruction complex. The fact that Rspo3 cannot rescue the action of Lrp6C-Cavla suggested that Rspo3 has the effect on the internalization of Lrp6 signal. The following coimmunoprecipitation experiment confirmed the hypothesis. Active Lrp6 signal can interacted with Caveolin 1a and subsequently transduced the canonical Wnt signal into cytoplasm in zebrfish embryos. However, co-injection with rspo3 reduced the interaction of active Lrp6 and Caveolin 1a, and therefore blocked the Wnt signal and inhibited canonical Wnt pathway. At last, forced expression of human RSPO3 in zebrafish embryos led to dorsoanterior patterning, which demonstrated that human RSPO3 had the similar effect with zebrafish Rspo3 on early embryo development, and RSPO3/Rspo3 had conserved function in zebrafish.The study on zebrafish rspol demonstrates that Rspol regulates convergence and extension movements (CE movements) through inhibiting the non-canonical Wnt/PCP signaling pathway in zebrafish embryos. The protein structure analysis shown that zebrafish Rspol had typical domain consist, including a SP domain, two FU domains, and a TSP1 domain. Zebrafish rspo1 was maternal deposited and highly expressed during 0-4 hpf. It had a ubiquitous expression fashion in early embryo stages. Ectopic expression of rspo1 affected the CE movements, led to the increased angle between anterior and posterior (CE defects) at 12 hpf. Mutants experiment shown the FU domains were functional domain for Rspo1. Further examinations shown Rspo1 had no effect on the dorsoventral development in early embryos, this data indicated the CE defects caused by Rspo1 is not due to the regulation to dorsoventral patterning. Marker genes in situ confirmed the effect of Rspol on CE movements. Using the Wnt/PCP reporter as a read-out of pathway activity, we found that forced expression of rspo1 inhibited Wnt/PCP activities in a dose-dependent manner, while knockdown of rspol stimulated that. Knockdown of rspol increased the phosphorylated level of JNK2, which indicated knockdown of rspo1 promoted Wnt/PCP activity. We also examined the functional interaction between Rspol and non-canonical Wnt ligands. Low dose of Wnt ligand mRNA or low does of rspol MO injection had no effect on embryo development, however, it can obviously activated Wnt/PCP pathway when this two were injected together, and induced CE defects. These results demonstrated Rspol participates and negatively regulates Wnt/PCP signaling pathway, and subsequently affects CE movements in zebrafish embryos.In summary, we focused on zebrafish rspo3 gene, and firstly reveals that Rspo3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling pathway in vivo in vertebrate. The function of zebrafish Rspol in early embryo development was explored. We demonstrate Rspol regulates CE movements through inhibiting Wnt/PCP signaling pathway for the first time. All these results advance and enrich the understanding to RSPOs/Rspos family, and provide newly insight and direction for the function research. Additionally, more evidence is demonstrated for the clinical research as Rspol a therapeutic target. |
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