斑马鱼Rspo3基因对Wnt通路负调控机理的初步研究
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摘要
Wnt信号通路是生物体中最重要的信号通路之一,其在生物体中控制细胞的增值、分化以及形态发生。R-spondin(Rspo)家族是一个近年来新发现的蛋白家族,由4个分泌性蛋白(Rspo1-4)组成。研究报道Rspo蛋白在脊椎动物的发育过程中发挥重要的作用。其作用与经典Wnt信号通路的配体相似,都是通过激活β-catenin发挥其生物学活性。目前关于硬骨鱼类Rspo蛋白结构与功能的报道很少。因此本研究以斑马鱼为模型,对Rspo3对Wnt通路的调控作用及其在斑马鱼发育过程中的功能进行了初步的研究。
我们通过荧光素酶报告基因检测,发现Rspo3在斑马鱼体内可以拮抗由外源Wnt3a配体诱导的Wnt通路的激活,也可以抑制内源的Wnt通路;同时也可以拮抗外源Wnt3a配体诱导的Wnt通路直接靶基因boz、sp5l的表达。而斑马鱼Rspo3的敲降则可以激活Wnt通路,造成胚胎头部减小,眼部减小。另外我们通过结构域删除突变体的显微注射实验发现,Rspo3的Fu结构域对Wnt通路的负调控至关重要。最有意思的是,与以往对其他物种的研究报道不同,斑马鱼Rspo3并不是通过作用于LRP6的胞外结构来实现对Wnt通路的调控的,而是通过作用于胞内的β-catenin蛋白或β-catenin上游的信号传导因子抑或是其他膜受体来抑制Wnt通路的。至此我们发现了斑马鱼与其他脊椎动物所不同的Rspo3蛋白对Wnt通路的作用方式。
斑马鱼作为一种新兴的模式生物,其在基因和蛋白质的结构与功能上都表现出很高的保守性,在研究脊椎动物的生长发育、人类疾病模型的构建等方面有着重大的意义,而发现其与其他脊椎动物在信号传导方面的一些差异,将会帮助我们更迅速、更准确、更深入的将其与高等生物的生长发育相结合,为研究高等生物的生长发育提供准确有效的遗传学工具。
Wnt signaling pathway is one of the key signaling pathways which control cell proliferation, differentiation and morphogenesis in organism. The R-spondins (Rspos) are a recently described family of 4 secreted proteins (Rspo1-4). Reported activities for Rspo proteins include essential roles in vertebrate development. Their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both Rspo and canonical Wnt ligands could induce the activation ofβ-catenin. However, their structures and functions in teleost fish are poorly understood. Therefore, this study aims at zebrafish Rspo3, and make a preliminary study of its influence to the Wnt pathway and the function in the development of zebrafish.
Luciferase reporter gene is used to find that Rspo3 in zebrafish can inhibit the Wnt signaling pathway not only when activated by the exogenous Wnt3a ligands but in the endogenous of it. And it also can restrain the expression of boz and sp5l actived by exogenous Wnt3a ligand, which is reported as the downstream target gene of Wnt pathway. And then, morpholinos are used to knock down the zebrafish Rspo3 and find that the Wnt pathway is obviously activated, resulting in the phenomenon of decreased head and eyes. Furthermore, Rspo3 gene mutants are constructed which miss the Fu domain, TB domain, TSP domain and NLS domain respectively, finding that the Fu domain of Rspo3 has a strong inhibition to Wnt pathway using the micro-injection. And most interestingly, different from the other species reported in previous studies, Rspo3 in zebrafish does not interact the structure of LRP6 at the cell surface to achieve the regulation of Wnt pathway, but by acting on theβ-catenin protein or some signaling factors upstreaming theβ-catenin or the other membrane receptors to inhibit the Wnt pathway.
At this point, a different regulation mode of Wnt pathway by Rspo3 protein in zebrafish is found. As a new model organism, the zebrafish has shown a high conservation in structure and function of genes and proteins compared to other vertebrates. And has a great significance for the study of vertebrates in the growth and development, even in the construction of human disease model. So, confirming the differences between zebrafish and the higher organisms in signal transduction and other aspects will help us combine it to the growth and development of higher organisms well, and use it as an accurate and effective genetic tool more quickly, more accurately and more thoroughly.
我们通过荧光素酶报告基因检测,发现Rspo3在斑马鱼体内可以拮抗由外源Wnt3a配体诱导的Wnt通路的激活,也可以抑制内源的Wnt通路;同时也可以拮抗外源Wnt3a配体诱导的Wnt通路直接靶基因boz、sp5l的表达。而斑马鱼Rspo3的敲降则可以激活Wnt通路,造成胚胎头部减小,眼部减小。另外我们通过结构域删除突变体的显微注射实验发现,Rspo3的Fu结构域对Wnt通路的负调控至关重要。最有意思的是,与以往对其他物种的研究报道不同,斑马鱼Rspo3并不是通过作用于LRP6的胞外结构来实现对Wnt通路的调控的,而是通过作用于胞内的β-catenin蛋白或β-catenin上游的信号传导因子抑或是其他膜受体来抑制Wnt通路的。至此我们发现了斑马鱼与其他脊椎动物所不同的Rspo3蛋白对Wnt通路的作用方式。
斑马鱼作为一种新兴的模式生物,其在基因和蛋白质的结构与功能上都表现出很高的保守性,在研究脊椎动物的生长发育、人类疾病模型的构建等方面有着重大的意义,而发现其与其他脊椎动物在信号传导方面的一些差异,将会帮助我们更迅速、更准确、更深入的将其与高等生物的生长发育相结合,为研究高等生物的生长发育提供准确有效的遗传学工具。
Wnt signaling pathway is one of the key signaling pathways which control cell proliferation, differentiation and morphogenesis in organism. The R-spondins (Rspos) are a recently described family of 4 secreted proteins (Rspo1-4). Reported activities for Rspo proteins include essential roles in vertebrate development. Their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both Rspo and canonical Wnt ligands could induce the activation ofβ-catenin. However, their structures and functions in teleost fish are poorly understood. Therefore, this study aims at zebrafish Rspo3, and make a preliminary study of its influence to the Wnt pathway and the function in the development of zebrafish.
Luciferase reporter gene is used to find that Rspo3 in zebrafish can inhibit the Wnt signaling pathway not only when activated by the exogenous Wnt3a ligands but in the endogenous of it. And it also can restrain the expression of boz and sp5l actived by exogenous Wnt3a ligand, which is reported as the downstream target gene of Wnt pathway. And then, morpholinos are used to knock down the zebrafish Rspo3 and find that the Wnt pathway is obviously activated, resulting in the phenomenon of decreased head and eyes. Furthermore, Rspo3 gene mutants are constructed which miss the Fu domain, TB domain, TSP domain and NLS domain respectively, finding that the Fu domain of Rspo3 has a strong inhibition to Wnt pathway using the micro-injection. And most interestingly, different from the other species reported in previous studies, Rspo3 in zebrafish does not interact the structure of LRP6 at the cell surface to achieve the regulation of Wnt pathway, but by acting on theβ-catenin protein or some signaling factors upstreaming theβ-catenin or the other membrane receptors to inhibit the Wnt pathway.
At this point, a different regulation mode of Wnt pathway by Rspo3 protein in zebrafish is found. As a new model organism, the zebrafish has shown a high conservation in structure and function of genes and proteins compared to other vertebrates. And has a great significance for the study of vertebrates in the growth and development, even in the construction of human disease model. So, confirming the differences between zebrafish and the higher organisms in signal transduction and other aspects will help us combine it to the growth and development of higher organisms well, and use it as an accurate and effective genetic tool more quickly, more accurately and more thoroughly.
引文
Aoki, M., M. Mieda, et al. (2007). "R-spondin3 is required for mouse placental development." Dev Biol 301(1): 218-26.
Behrens, J., B. A. Jerchow, et al. (1998). "Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta." Science 280(5363): 596-9.
Bejsovec, A. (2005). "Wntpathway activation: new relations and locations." Cell 120(1): 11-4. Bell, S. M., C. M. Schreiner, et al. (2008). "R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis." Development 135(6): 1049-58.
Binnerts, M. E., K. A. Kim, et al. (2007). "R-Spondin1 regulates Wntsignaling by inhibiting internalization of LRP6." Proc Natl Acad Sci U S A 104(37): 14700-5.
Blaydon, D. C., Y. Ishii, et al. (2006). "The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wntsignaling, is mutated in inherited anonychia." Nat Genet 38(11): 1245-7.
Chadi, S., L. Buscara, et al. (2009). "R-spondin1 is required for normal epithelial morphogenesis during mammary gland development." Biochem Biophys Res Commun 390(3): 1040-3.
Chen, J. Z., S. Wang, et al. (2002). "Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR." Mol Biol Rep 29(3): 287-92.
Clevers, H. (2006). "Wnt/beta-catenin signaling in development and disease." Cell 127(3): 469-80. Davidson, G., W. Wu, et al. (2005). "Casein kinase 1 gamma couples Wntreceptor activation to cytoplasmic signal transduction." Nature 438(7069): 867-72.
Ellwanger, K., H. Saito, et al. (2008). "Targeted disruption of the Wntregulator Kremen induces limb defects and high bone density." Mol Cell Biol 28(15): 4875-82.
Gordon, M. D. and R. Nusse (2006). "Wntsignaling: multiple pathways, multiple receptors, and multiple transcription factors." J Biol Chem 281(32): 22429-33.
Kamata, T., K. Katsube, et al. (2004). "R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants." Biochim Biophys Acta 1676(1): 51-62.
Kazanskaya, O., A. Glinka, et al. (2004). "R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis." Dev Cell 7(4): 525-34.
Kazanskaya, O., B. Ohkawara, et al. (2008). "The Wntsignaling regulator R-spondin 3 promotes angioblast and vascular development." Development 135(22): 3655-64.
Kim, K. A., M. Kakitani, et al. (2005). "Mitogenic influence of human R-spondin1 on the intestinal epithelium." Science 309(5738): 1256-9.
Kim, K. A., M. Wagle, et al. (2008). "R-Spondin family members regulate the Wntpathway by a common mechanism." Mol Biol Cell 19(6): 2588-96.
Kim, K. A., J. Zhao, et al. (2006). "R-Spondin proteins: a novel link to beta-catenin activation." Cell Cycle 5(1): 23-6.
Kirikoshi, H., H. Sekihara, et al. (2001). "Expression profiles of 10 members of Frizzled gene family in human gastric cancer." Int J Oncol 19(4): 767-71.
Kishida, S., H. Yamamoto, et al. (1998). "Axin, a negative regulator of the Wntsignaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin." J Biol Chem 273(18): 10823-6.
Liu, C., Y. Li, et al. (2002). "Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism." Cell 108(6): 837-47.
Lu, W., K. A. Kim, et al. (2008). "R-spondin1 synergizes with Wnt3A in inducing osteoblast differentiation and osteoprotegerin expression." FEBS Lett 582(5): 643-50.
MacDonald, B. T., K. Tamai, et al. (2009). "Wnt/beta-catenin signaling: components, mechanisms, and diseases." Dev Cell 17(1): 9-26.
Mathew, L. K., S. S. Sengupta, et al. (2008). "Crosstalk between AHR and Wntsignaling through R-Spondin1 impairs tissue regeneration in zebrafish." FASEB J 22(8): 3087-96.
Munroe, D. G., A. K. Gupta, et al. (1999). "Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2." Proc Natl Acad Sci U S A 96(4): 1569-73.
Nam, J. S., T. J. Turcotte, et al. (2006). "Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression." J Biol Chem 281(19): 13247-57.
Nam, J. S., T. J. Turcotte, et al. (2007). "Dynamic expression of R-spondin family genes in mouse development." Gene Expr Patterns 7(3): 306-12.
Parma, P., O. Radi, et al. (2006). "R-spondin1 is essential in sex determination, skin differentiation and malignancy." Nat Genet 38(11): 1304-9.
Sher, I., B. K. Yeh, et al. (2003). "Structure-based mutational analyses in FGF7 identify new residues involved in specific interaction with FGFR2IIIb." FEBS Lett 552(2-3): 150-4.
Tamai, K., X. Zeng, et al. (2004). "A mechanism for Wntcoreceptor activation." Mol Cell 13(1): 149-56.
Tolwinski, N. S. and E. Wieschaus (2004). "A nuclear function for armadillo/beta-catenin." PLoS Biol 2(4): E95.
Wei, Q., C. Yokota, et al. (2007). "R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling." J Biol Chem 282(21): 15903-11.
Willert, K., S. Shibamoto, et al. (1999). "Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex." Genes Dev 13(14): 1768-73.
Zeng, X., H. Huang, et al. (2008). "Initiation of Wntsignaling: control of Wntcoreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions." Development 135(2): 367-75.
Zhao, J., K. A. Kim, et al. (2009). "R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/beta-catenin pathway." Proc Natl Acad Sci U S A 106(7): 2331-6.
Behrens, J., B. A. Jerchow, et al. (1998). "Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta." Science 280(5363): 596-9.
Bejsovec, A. (2005). "Wntpathway activation: new relations and locations." Cell 120(1): 11-4. Bell, S. M., C. M. Schreiner, et al. (2008). "R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis." Development 135(6): 1049-58.
Binnerts, M. E., K. A. Kim, et al. (2007). "R-Spondin1 regulates Wntsignaling by inhibiting internalization of LRP6." Proc Natl Acad Sci U S A 104(37): 14700-5.
Blaydon, D. C., Y. Ishii, et al. (2006). "The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wntsignaling, is mutated in inherited anonychia." Nat Genet 38(11): 1245-7.
Chadi, S., L. Buscara, et al. (2009). "R-spondin1 is required for normal epithelial morphogenesis during mammary gland development." Biochem Biophys Res Commun 390(3): 1040-3.
Chen, J. Z., S. Wang, et al. (2002). "Cloning and identification of a cDNA that encodes a novel human protein with thrombospondin type I repeat domain, hPWTSR." Mol Biol Rep 29(3): 287-92.
Clevers, H. (2006). "Wnt/beta-catenin signaling in development and disease." Cell 127(3): 469-80. Davidson, G., W. Wu, et al. (2005). "Casein kinase 1 gamma couples Wntreceptor activation to cytoplasmic signal transduction." Nature 438(7069): 867-72.
Ellwanger, K., H. Saito, et al. (2008). "Targeted disruption of the Wntregulator Kremen induces limb defects and high bone density." Mol Cell Biol 28(15): 4875-82.
Gordon, M. D. and R. Nusse (2006). "Wntsignaling: multiple pathways, multiple receptors, and multiple transcription factors." J Biol Chem 281(32): 22429-33.
Kamata, T., K. Katsube, et al. (2004). "R-spondin, a novel gene with thrombospondin type 1 domain, was expressed in the dorsal neural tube and affected in Wnts mutants." Biochim Biophys Acta 1676(1): 51-62.
Kazanskaya, O., A. Glinka, et al. (2004). "R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis." Dev Cell 7(4): 525-34.
Kazanskaya, O., B. Ohkawara, et al. (2008). "The Wntsignaling regulator R-spondin 3 promotes angioblast and vascular development." Development 135(22): 3655-64.
Kim, K. A., M. Kakitani, et al. (2005). "Mitogenic influence of human R-spondin1 on the intestinal epithelium." Science 309(5738): 1256-9.
Kim, K. A., M. Wagle, et al. (2008). "R-Spondin family members regulate the Wntpathway by a common mechanism." Mol Biol Cell 19(6): 2588-96.
Kim, K. A., J. Zhao, et al. (2006). "R-Spondin proteins: a novel link to beta-catenin activation." Cell Cycle 5(1): 23-6.
Kirikoshi, H., H. Sekihara, et al. (2001). "Expression profiles of 10 members of Frizzled gene family in human gastric cancer." Int J Oncol 19(4): 767-71.
Kishida, S., H. Yamamoto, et al. (1998). "Axin, a negative regulator of the Wntsignaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin." J Biol Chem 273(18): 10823-6.
Liu, C., Y. Li, et al. (2002). "Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism." Cell 108(6): 837-47.
Lu, W., K. A. Kim, et al. (2008). "R-spondin1 synergizes with Wnt3A in inducing osteoblast differentiation and osteoprotegerin expression." FEBS Lett 582(5): 643-50.
MacDonald, B. T., K. Tamai, et al. (2009). "Wnt/beta-catenin signaling: components, mechanisms, and diseases." Dev Cell 17(1): 9-26.
Mathew, L. K., S. S. Sengupta, et al. (2008). "Crosstalk between AHR and Wntsignaling through R-Spondin1 impairs tissue regeneration in zebrafish." FASEB J 22(8): 3087-96.
Munroe, D. G., A. K. Gupta, et al. (1999). "Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2." Proc Natl Acad Sci U S A 96(4): 1569-73.
Nam, J. S., T. J. Turcotte, et al. (2006). "Mouse cristin/R-spondin family proteins are novel ligands for the Frizzled 8 and LRP6 receptors and activate beta-catenin-dependent gene expression." J Biol Chem 281(19): 13247-57.
Nam, J. S., T. J. Turcotte, et al. (2007). "Dynamic expression of R-spondin family genes in mouse development." Gene Expr Patterns 7(3): 306-12.
Parma, P., O. Radi, et al. (2006). "R-spondin1 is essential in sex determination, skin differentiation and malignancy." Nat Genet 38(11): 1304-9.
Sher, I., B. K. Yeh, et al. (2003). "Structure-based mutational analyses in FGF7 identify new residues involved in specific interaction with FGFR2IIIb." FEBS Lett 552(2-3): 150-4.
Tamai, K., X. Zeng, et al. (2004). "A mechanism for Wntcoreceptor activation." Mol Cell 13(1): 149-56.
Tolwinski, N. S. and E. Wieschaus (2004). "A nuclear function for armadillo/beta-catenin." PLoS Biol 2(4): E95.
Wei, Q., C. Yokota, et al. (2007). "R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling." J Biol Chem 282(21): 15903-11.
Willert, K., S. Shibamoto, et al. (1999). "Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex." Genes Dev 13(14): 1768-73.
Zeng, X., H. Huang, et al. (2008). "Initiation of Wntsignaling: control of Wntcoreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions." Development 135(2): 367-75.
Zhao, J., K. A. Kim, et al. (2009). "R-Spondin1 protects mice from chemotherapy or radiation-induced oral mucositis through the canonical Wnt/beta-catenin pathway." Proc Natl Acad Sci U S A 106(7): 2331-6.