Rspo1调控斑马鱼胚胎汇聚延伸运动的研究

Rspo1 (R-spondin 1)是分泌型Rspos (R-spondins)蛋白家族的成员,在雌性发育、血管生成和癌症等多个方面具有调控作用。为了研究Rspo1在早期胚胎发育中的功能,以斑马鱼(Danio rerio)作为模式生物,利用反转录PCR及原位杂交技术检测rspo1基因的时空表达模式;通过显微注射rspo1 mRNA或rspo1反义寡核苷酸(Morpholino, MO)对rspo1进行过表达或敲降;通过形态观察及原位杂交技术检测胚胎汇聚延伸(Convergence and extension, CE)运动是否正常;利用荧光素酶活性检测实验测定Wnt/PCP信号通路活性水平;通过蛋白印迹法检测表征Wnt/PCP信号通路活性的磷酸化JNK (Jun N-terminal kinase)蛋白的水平。结果显示:rspo1为母源基因,在12hpf前胚胎中呈全身性表达, rspo1的过表达或敲降均影响胚胎的CE运动;过表达rspo1降低Wnt/PCP信号通路报告质粒的活性,而敲降rspo1则增加其活性,与之相一致, rspo1敲降的胚胎中磷酸化JNK的水平显著升高;此外, rsp...     

The Wnt signaling regulator R-spondin 3 promotes angioblast and vascular development

The vertebrate embryonic vasculature develops from angioblasts, which are specified from mesodermal precursors and develop in close association with blood cells. The signals that regulate embryonic vasculogenesis and angiogenesis are incompletely understood. Here, we show that R-spondin 3 (Rspo3), a member of a novel family of secreted proteins in vertebrates that activate Wnt/beta-catenin signaling, plays a key role in these processes. In Xenopus embryos, morpholino antisense knockdown of Rspo3 induces vascular defects because Rspo3 is essential for regulating the balance between angioblast and blood cell specification. In mice, targeted disruption of Rspo3 leads to embryonic lethality caused by vascular defects. Specifically in the placenta, remodeling of the vascular plexus is impaired. In human endothelial cells, R-spondin signaling promotes proliferation and sprouting angiogenesis in vitro, indicating that Rspo3 can regulate endothelial cells directly. We show that vascular endothelial growth factor is an immediate early response gene and a mediator of R-spondin signaling. The results identify Rspo3 as a novel, evolutionarily conserved angiogenic factor in embryogenesis.     

R-spondin1 synergizes with Wnt3A in inducing osteoblast differentiation and osteoprotegerin expression

R-spondins are a new group of Wnt/beta-catenin signaling agonists, however, the role of these proteins in bone remains unclear. We reported herein that R-sponin1 (Rspo1) acted synergistically with Wnt3A to activate Wnt/beta-catenin signaling in the uncommitted mesenchymal C2C12 cells. Furthermore, we found that Rspo1 at concentrations as low as 10 ng/ml synergized strongly with Wnt3A to induce C2C12 osteoblastic differentiation and osteoprotegerin expression. These events were blocked by Wnt/beta-catenin signaling antagonist Dickkopf-1. Finally, we demonstrated that Rspo1 synergized with Wnt3A to induce primary mouse osteoblast differentiation. Together, these findings suggest that Rpos1 may play an important role in bone remodeling.     

Rspo2/Int7 regulates invasiveness and tumorigenic properties of mammary epithelial cells

Rspo2 was identified as a novel common integration site (CIS) for the mouse mammary tumor virus (MMTV) in viral induced mouse mammary tumors. Here we show that Rspo2 modulates Wnt signaling in mouse mammary epithelial cells. Co‐expression of both genes resulted in an intermediate growth phenotype on plastic and had minor effects on the growth‐promoting properties of Wnt1 in soft agar. However, individual Rspo2 and Wnt1 HC11 transfectants as well as the double transfectant were tumorigenic in athymic nude mice, with tumors from each line having distinctive histological characteristics. Rspo2 and Rspo2/Wnt1 tumors contained many spindle cells, consistent with an epithelial–mesenchymal transformation (EMT) phenotype. When Rspo2 and Rspo2/Wnt1 tumor cells were transferred into naïve mice, they exhibited greater metastatic activity than cells derived from Wnt1 tumors. For comparison, C57MG/Wnt1/Rspo2 co‐transfectants exhibited invasive properties in three‐dimensional (3D) Matrigel cultures that were not seen with cells transfected only with Wnt1 or Rspo2. Use of Dickkopf‐1, a specific antagonist of the Wnt/β‐catenin pathway, or short hairpin RNA targeting β‐catenin expression demonstrated that the invasive activity was not mediated by β‐catenin. Our results indicate that Rspo2 and Wnt1 have mutually distinct effects on mammary epithelial cell growth and these effects are context‐dependent. While Rspo2 and Wnt1 act synergistically in the β‐catenin pathway, other mechanisms are responsible for the invasive properties of stable double transfectants observed in 3D Matrigel cultures. J. Cell. Physiol. 227: 1960–1971, 2012. © 2011 Wiley Periodicals, Inc.     

A conditional allele of Rspo3 reveals redundant function of R‐spondins during mouse limb development

Summary: R‐spondins are secreted ligands that bind cell surface receptors and activate Wnt/β‐catenin signaling. Human mutations and gene inactivation studies in mice have revealed a role for these four proteins (RSPO1‐4) in diverse developmental processes ranging from sex determination to limb development. Among the genes coding for R‐spondins, only inactivation of Rspo3 shows early embryonic lethality (E10.5 in mice). Therefore, a conditional allele of this gene is necessary to understand the function of R‐spondins throughout murine development. To address this need, we have produced an allele in which loxP sites flank exons 2–4 of Rspo3, allowing tissue‐specific deletion of these exons in the presence of Cre recombinase. We used these mice to investigate the role of Rspo3 during limb development and found that limbs ultimately developed normally in the absence of Rspo3 function. However, severe hindlimb truncations resulted when Rspo3 and Rspo2 mutations were combined, demonstrating redundant function of these genes. genesis 50:741–749, 2012. © 2012 Wiley Periodicals, Inc.     

Craniofacial malformation in R-spondin2 knockout mice

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/β-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/β-catenin signaling, Rspo2, we generated and characterized Rspo2^−/− mice. We found CLP with mild facial skeletal defects in Rspo2^−/− mice. Additionally, Rspo2^−/− mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/β-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2^−/− mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/β-catenin signaling.     

R-Spondin2 is a secreted activator of Wnt/beta-catenin signaling and is required for Xenopus myogenesis

We have carried out a small pool expression screen for modulators of the Wnt/beta-catenin pathway and identified Xenopus R-spondin2 (Rspo2) as a secreted activator of this cascade. Rspo2 is coexpressed with and positively regulated by Wnt signals and synergizes with Wnts to activate beta-catenin. Analyses of functional interaction with components of the Wnt/beta-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/beta-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-beta pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/beta-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/beta-catenin cascade.     

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function

R-spondins (Rspos) potentiate Wnt/β-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein, demonstrating for the first time specific amino acid residues in Rspos that are critical for β-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide-bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a β-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.     

Wnt11 Promotes Osteoblast Maturation and Mineralization through R-spondin 2

Wnt11 signals through both canonical (β-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases β-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/β-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through β-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b enhances osteoblast differentiation (1). Canonical Wnt signaling through β-catenin has also been shown to enhance the chondroinductive and osteoinductive properties of BMP2² (2, 3). During BMP2-induced osteoblast differentiation of mesenchymal stem cell lines, cross-talk between BMP and Wnt pathways converges through the interaction of Smad4 with β-catenin (2).Canonical Wnt signaling is also critical for skeletal development and homeostasis. During limb development, expression of Wnt3a in the apical ectodermal ridge of limb buds maintains cells in a highly proliferative and undifferentiated state (4, 5). Disruption of canonical Wnt signaling in Lrp5/Lrp6 compound knock-out mice results in limb- and digit-patterning defects (6). Wnt signaling is also involved in the maintenance of post-natal bone mass. Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass, whereas loss of Lrp5 function is associated with decreased bone mass and osteoporosis pseudoglioma syndrome (7, 8). Mice with increased Wnt10b expression have increased trabecular bone, whereas Wnt10b-deficient mice have reduced trabecular bone (9). Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased trabecular bone accrual throughout adulthood (10).Although canonical Wnt signaling regulates osteoblastogenesis and bone formation, the profile of endogenous Wnts that play a role in osteoblast differentiation and maturation is not well described. During development, Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum (11). Wnt11 expression is increased during glucocorticoid-induced osteogenesis (12), indicating a potential role for Wnt11 in osteoblast differentiation. Interestingly, Wnt11 activates both β-catenin-dependent as well as β-catenin-independent signaling pathways (13). Targeted disruption of Wnt11 results in late embryonic/early post-natal death because of cardiac dysfunction (14). Although these mice have no reported skeletal developmental abnormalities, early lethality obfuscates a detailed examination of post-natal skeletal modeling and remodeling.In murine development, Wnt11 expression overlaps with the expression of R-spondin 2 (Rspo2) in the apical ectodermal ridge (11, 15). R-spondins are a novel family of proteins that share structural features, including two conserved cysteinerich furin-like domains and a thrombospondin type I repeat (16). The four R-spondin family members can activate canonical Wnt signaling (15, 17–19). Rspo3 interacts with Frizzled 8 and Lrp6 and enhances Wnt ligand signaling. Rspo1 enhances Wnt signaling by interacting with Lrp6 and inhibiting Dkk-mediated receptor internalization (20). Rspo1 was also shown to potentiate Wnt3a-mediated osteoblast differentiation (21). Rspo2 knock-out mice, which die at birth, have limb patterning defects associated with altered β-catenin signaling (22–24). However, the role of Rspo2 in osteoblast differentiation and maturation remains unclear.Herein we report that Wnt11 overexpression in MC3T3E1 pre-osteoblasts activates β-catenin and augments BMP-induced osteoblast maturation and mineralization. Wnt11 increases the expression of Rspo2. Overexpression of Rspo2 in MC3T3E1 is sufficient for augmenting BMP-induced osteoblast maturation and mineralization. Although antagonism of Tcf/β-catenin signaling blocks the osteogenic effects of Wnt11, Rspo2 rescues this block, and knockdown of Rspo2 shows that it is required for Wnt11-mediated osteoblast maturation and mineralization. These studies identify both Wnt11 and Rspo2 as novel mediators of osteoblast maturation and mineralization.     

Accelerated Coronary Angiogenesis by Vegfr1-Knockout Endocardial Cells

During mouse heart development, ventricular endocardial cells give rise to the coronary arteries by angiogenesis. Myocardially-derived vascular endothelial growth factor-a (Vegfa) regulates embryonic coronary angiogenesis through vascular endothelial growth factor-receptor 2 (Vegfr2) expressed in the endocardium. In this study, we investigated the role of endocardially-produced soluble Vegfr1 (sVegfr1) in the coronary angiogenesis. We deleted sVegfr1 in the endocardium of the developing mouse heart and found that this deletion resulted in a precocious formation of coronary plexuses. Using an ex vivo coronary angiogenesis assay, we showed that the Vegfr1-null ventricular endocardial cells underwent excessive angiogenesis and generated extensive endothelial tubular networks. We also revealed by qPCR analysis that expression of genes involved in the Vegf-Notch pathway was augmented in the Vegfr1-null hearts. We further showed that inhibition of Notch signaling blocked the formation of coronary plexuses by the ventricular endocardial cells. These results establish that Vegfr1 produced in the endocardium negatively regulates embryonic coronary angiogenesis, possibly by limiting the Vegf-Notch signaling.     

R-spondin 2 is required for normal laryngeal-tracheal, lung and limb morphogenesis

Herein, we demonstrate that Lrp6-mediated R-spondin 2 signaling through the canonical Wnt pathway is required for normal morphogenesis of the respiratory tract and limbs. We show that the footless insertional mutation creates a severe hypomorphic R-spondin 2 allele (Rspo2(Tg)). The predicted protein encoded by Rspo2(Tg) neither bound the cell surface nor activated the canonical Wnt signaling reporter TOPFLASH. Rspo2 activation of TOPFLASH was dependent upon the second EGF-like repeat of Lrp6. Rspo2(Tg/Tg) mice had severe malformations of laryngeal-tracheal cartilages, limbs and palate, and lung hypoplasia consistent with sites of Rspo2 expression. Rspo2(Tg/Tg) lung defects were associated with reduced branching, a reduction in TOPGAL reporter activity, and reduced expression of the downstream Wnt target Irx3. Interbreeding the Rspo2(Tg) and Lrp6(-) alleles resulted in more severe defects consisting of marked lung hypoplasia and absence of tracheal-bronchial rings, laryngeal structures and all limb skeletal elements.     

Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis

1. Download : Download high-res image (218KB)
2. Download : Download full-size image

Highlights? Discovery that Rspo3 binds syndecan 4 ? Rspo3/Sdc4 induce Wnt/PCP signaling via clathrin-mediated endocytosis ? Rspo3/PCP signaling is essential for gastrulation and cartilage morphogenesis     

Mouse R-spondin2 is required for apical ectodermal ridge maintenance in the hindlimb

The R-spondin (Rspo) family of proteins consists of secreted cysteine-rich proteins that can activate β-catenin signaling via the Frizzled/LRP5/6 receptor complex. Here, we report that targeted inactivation of the mouse Rspo2 gene causes developmental limb defects, especially in the hindlimb. Although the initiation of the expression of apical ectodermal ridge (AER)-specific genes, including fibroblast growth factor 8 (FGF8) and FGF4 occurred normally, the maintenance of these marker expressions was significantly defective in the hindlimb of Rspo2(−/−) mice. Consistent with the ligand role of R-spondins in the Wnt/β-catenin signaling pathway, expression of Axin2 and Sp8, targets for β-catenin signaling, within AER was greatly reduced in Rspo2(−/−) embryos. Furthermore, sonic hedgehog (Shh) signaling within the hindlimbs of Rspo2(−/−) mice was also significantly decreased. Rspo2 is expressed in the AER of all limb buds, however the stunted phenotype is significantly more severe in the hindlimbs than the forelimbs and strongly biased to the left side. Our findings strongly suggest that Rspo2 expression in the AER is required for AER maintenance likely by regulating Wnt/β-catenin signaling.     

Wnt/beta-catenin signaling controls Mespo expression to regulate segmentation during Xenopus somitogenesis

The vertebral column is derived from somites, which are transient segments of the paraxial mesoderm that are present in developing vertebrates. The strict spatial and temporal regulation of somitogenesis is of crucial developmental importance. Signals such as Wnt and FGF play roles in somitogenesis, but details regarding how Wnt signaling functions in this process remain unclear. In this study, we report that Wnt/beta-catenin signaling regulates the expression of Mespo, a basic-helix-loop-helix (bHLH) gene critical for segmental patterning in Xenopus somitogenesis. Transgenic analysis of the Mespo promoter identifies Mespo as a direct downstream target of Wnt/beta-catenin signaling pathway. We also demonstrate that activity of Wnt/beta-catenin signaling in somitogenesis can be enhanced by the PI3-K/AKT pathway. Our results illustrate that Wnt/beta-catenin signaling in conjunction with PI3-K/AKT pathway plays a key role in controlling development of the paraxial mesoderm.     

Rspo1对雌性脊椎动物性别分化的功能

长期以来雌性脊椎动物的性别分化被认为是一个“默认”的程序.但是近些年研究发现,Rspo1基因的突变或缺失可导致哺乳动物XX型个体性反转为雄性.Rspo1在鱼类、两栖爬行类、鸟类和哺乳类动物性腺发育的不同阶段表达,其表达在雌雄个体性别分化时期有差异,是潜在的性别调控基因.Rspo1在性别发育早期可通过Wnt/β-catenin信号通路调控性腺分化相关因子的表达,影响原始生殖细胞分裂增殖、细胞周期和生长发育,参与调控性腺中体细胞的分化.本文总结了近年来Rspo1在脊椎动物中的表达调控及其在雌性性别决定方面功能的研究进展.