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Noncanonical Wnt signals control morphogenetic movements during vertebrate gastrulation. Casein kinase I epsilon (CKIvarepsilon) is a Wnt-regulated kinase that regulates Wnt/beta-catenin signaling and has a beta-catenin-independent role(s) in morphogenesis that is poorly understood. Here we report the identification of a CKIvarepsilon binding partner, SIPA1L1/E6TP1, a GAP (GTPase activating protein) of the Rap small GTPase family. We show that CKIvarepsilon phosphorylates SIPA1L1 to reduce its stability and thereby increase Rap1 activation. Wnt-8, which activates CKIvarepsilon, enhances the CKIvarepsilon-dependent phosphorylation and degradation of SIPA1L1. In early Xenopus or zebrafish development, inactivation of the Rap1 pathway results in abnormal gastrulation and a shortened anterior-posterior axis. Although CKIvarepsilon also transduces Wnt/beta-catenin signaling, inhibition of Rap1 does not alter beta-catenin-regulated gene expression. Our data demonstrate a role for CKIvarepsilon in noncanonical Wnt signaling and indicate that Wnt regulates morphogenesis in part through CKIvarepsilon-mediated control of Rap1 signaling.
Figure 4. Rap1 Is Required for Wnt-8- and CKI-Mediated Axis Development(A) Expression of DN-Rap1 in Xenopus embryos causes an open yolk plug at the tail region of the presumptive secondary axis. mRNA of Wnt-8 (5 pg) or CKI (1 ng) was coinjected with DN-Rap1 (100 pg) into a single ventral cell of four-cell stage embryos. Embryos were scored and photographed at stage 18. Whole-mount in situ hybridization for Sox-2 expression was performed on stage 14 embryos. Black arrowhead, double axis; red arrowhead, gastrulation defect; bottom, scoring of stage 18 embryos.(B) Dorsal expression of DN-Rap1 alone causes a gastrulation defect in Xenopus embryos, whereas ventral expression does not affect gastrulation in the majority of embryos. DN-Rap1 RNA (500 pg) was injected into a single dorsal or ventral cell of four-cell stage embryos. Embryos were photographed at stages 18 and 30 as indicated. Black arrowhead, normal axis; red arrowhead, blastopore closure defect.(C) Disruption of Rap1 activity inhibits activin-induced convergent extension of animal cap. Convergent extension assays were performed on the Xenopus embryos, dorsally injected with RNA encoding GFP, DN-Rap1, or SIPA1L1(Cδ). Animal caps were photographed when the sibling embryos reached stage 18. The length-to-width ratios of the activin-treated animal caps were measured with MetaMorph software and are shown in the lower-right panel. Individual results for each animal cap are denoted by , and the average is shown at the top and denoted by — in the graph.(D) Xenopus embryos injected with Rap1 MO display a defect in gastrulation. Black arrowhead, normal axis; red arrowhead, defect in blastopore closure. The CE defect could be rescued by wild-type (WT) hRap1. Control MO (40 ng) or MOs against XRap1A (40 ng) and XRap1B (40 ng) were injected together into the animal cap of Xenopus at the one-cell stage. Two to four picograms of HA-tagged hRap1 were injected into two dorsal cells at the four-cell stage to rescue the Rap1-MO-caused defects. Only 5% (n = 99) of the embryos exhibit gastrulation defects after rescue. Red arrowhead, blastopore closure defect.
Figure 5. CKI and SIPA1L1 Are Upstream of Rap1(A) Dorsal expression of SIPA1L1(FL) or SIPA1L1(Cδ) alone in Xenopus embryos causes a gastrulation defect in 23% (n = 65) and 73% (n = 70) of embryos, respectively. Ventral expression does not induce an obvious phenotype in the majority of injected embryos (95% and 79% unaffected, n = 39 and 38, respectively). Four-cell stage embryos were injected with 1 ng of RNAs encoding SIPA1L1(FL) or SIPA1L1(Cδ) and photographed at stages 18 and 30. Black arrowhead, normal axis; red arrowhead, blastopore closure defect.(B) SIPA1L1(Cδ) but not SIPA1L1(FL) inhibits gastrulation at the presumptive secondary axis. (i) Uninjected control. (ii–iv) Xenopus four-cell stage embryos were injected dorsally with mRNA encoding (ii) CKI (1 ng) + GFP (250 pg); (iii) CKI + SIPA1L1(FL) (250 pg); and (iv) CKI + SIPA1L1(Cδ) (250 pg). (v) CKI + SIPA1L1(Cδ, mtGAP) (250 pg). In situ hybridization for Sox-2 expression was performed in stage 15 embryos. Black arrowhead, double axis; red arrowhead, gastrulation defect. Scoring data of stage 18 embryos are shown in the lower-right panel.
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