1. Volume 12, Issue 3, Pages 335-347 (March 2007)
A Wnt-CKIɛ-Rap1 Pathway Regulates Gastrulation by Modulating SIPA1L1, a Rap GTPase Activating Protein 
I-Chun Tsai, Jeffrey D. Amack, Zhong-Hua Gao, Vimla Band, H. Joseph Yost, David M. Virshup 
Developmental Cell 
Volume 12, Issue 3, Pages (March 2007)
DOI: /j.devcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1 CKIɛ Binds to SIPA1L1, a GAP of Rap Small GTPase
(A) In vivo interaction of SIPA1L1 and CKIɛ. Lanes 1–6 demonstrate the reciprocal coimmunoprecipitation (IP) of Myc-tagged SIPA1L1 and HA-tagged CKIɛ in HEK293 cells. Lanes 7–12 show immunoblots (IB) of whole-cell lysates demonstrating the expression of Myc-SIPA1L1 and HA-CKIɛ. CKIɛ(WT), wild-type CKIɛ; CKIɛ(KD), a kinase dead K38R mutant.
(B) SIPA1L1 binds to endogenous CKIɛ. Myc-SIPA1L1 was expressed in HEK293 cells and immunoprecipitated, followed by immunoblotting with a CKIɛ-specific Ab. GFP was expressed as a negative control.
(C) The carboxyl terminus of SIPA1L1 is required for interaction with CKIɛ. Upper and middle panels, co-IP of Myc-tagged SIPA1L1 truncated constructs and 3× flag-tagged CKIɛ. Bottom panel, expression of SIPA1L1 constructs. The figure is from a single exposure of the immunoblot; the lanes have been reordered for clarity.
(D) In vitro binding of SIPA1L1 to CKIɛ. Deletion constructs of SIPA1L1 were expressed in reticulocyte lysates. Recombinant 6× His-tagged CKIɛ was added where indicated. Luciferase was a negative control. In, input; 20% of the amount used for binding assay.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
CKIɛ Phosphorylates SIPA1L1, Promotes Its Degradation, and Alleviates SIPA1L1-Mediated Rap1 Inhibition
(A) CKIɛ expression causes an electrophoretic mobility shift of SIPA1L1(FL) but not SIPA1L1(CΔ). IB, immunoblot of whole-cell lysates from HEK293 cells expressing the indicated proteins. CKIɛ and α-tubulin abundance are shown in the middle and bottom panels.
(B) SIPA1L1 is phosphorylated by CKIɛ. Myc-SIPA1L1 plus CKIɛ or GFP (as the negative control for IP) were expressed in HEK293 cells. Myc-SIPA1L1 was precipitated and treated with or without calf intestinal alkaline phosphatase (CIP).
(C) CKIɛ stimulates SIPA1L1 degradation. Lanes 1–5, Myc-SIPA1L1 coexpressed with CKIɛ(WT) or CKIɛ(KD) as indicated. Lanes 5 and 6, cells expressing Myc-SIPA1L1 were treated with IC261 (50 μM) for 5 hr. IC261 treatment increased SIPA1L1 abundance (compare lane 5 to lane 3 and lane 6 to lane 2). Expression of CKIɛ(WT/KD) and endogenous α-tubulin abundance are shown in middle and lower panels.
(D) CKIɛ rescues SIPA1L1-mediated Rap1 inhibition. Myc-SIPA1L1(FL) or Myc-SIPA1L1(CΔ) was coexpressed with or without CKIɛ in the CHO cells, followed by GST-RalGDS pull-down assay. The abundance of GTP-Rap1 and total Rap1 was quantified by ImageQuant. The ratio of GTP-Rap1 to total Rap1 is shown in the lower panel.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
Wnt-8 Regulates CKIɛ-Dependent Phosphorylation and Abundance of SIPA1L1
(A) Wnt-8 stimulates CKIɛ-dependent phosphorylation of SIPA1L1. Protein two-dimensional electrophoresis of Myc-SIPA1L1 was performed after treatment of HEK293 cells with (i) control medium, (ii) Wnt-8 conditioned medium, or (iii) Wnt-8 conditioned medium plus IC261. Arrows indicate Myc-SIPA1L1 with several different isoelectric points.
(B) Wnt-8 conditioned medium reduces the abundance of SIPA1L1. Upper panel, after 6 hr of conditioned medium incubation and IC261 treatment, immunoblot analysis of Myc-SIPA1L1 was performed. Lower panel, endogenous Dvl-1 abundance (as a loading control).
(C) Wnt-8 conditioned medium increases Rap1 activation, whereas IC261 blocks basal and Wnt-8-mediated Rap1 activation. HEK293 cells were incubated with control or Wnt-8 conditioned medium and treated with or without addition of IC261 as indicated. After 6 hr, cells were lysed, followed by GST-RalGDS pull-down assay. Quantification of each lane was performed as described in Figure 2D and shown in the right panel.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Rap1 Is Required for Wnt-8- and CKIɛ-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 CKIɛ (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.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5 CKIɛ 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) CKIɛ (1 ng) + GFP (250 pg); (iii) CKIɛ + SIPA1L1(FL) (250 pg); and (iv) CKIɛ + SIPA1L1(CΔ) (250 pg). (v) CKIɛ + 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.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6 Regulation of Rap1 GTPase in Zebrafish Development
(A) Injection of DN-Rap1 RNA, SIPA1L1(CΔ) RNA, or Rap1B MO in zebrafish embryos results in a bent and shortened AP axis. RNAs encoding GFP (500 pg; as negative control), DN-Rap1 (500 pg), or SIPA1L1(CΔ) (250 pg) were injected into zebrafish embryos at the one- to four-cell stages. Between 2 and 3 dpf, 42% (n = 150) of DN-Rap1-, 37% (n = 237) of SIPA1L1(CΔ)-, and 58% (n = 63) of Rap1B-MO-injected embryos displayed a shortened and bent AP axis that varied in severity. AP defects were seen in only 6% (n = 143) of GFP-injected embryos. Embryos injected with either 2, 2.5, or 3.4 ng of Rap1B MO showed similar phenotypes and were pooled for analysis.
(B) Downregulation of Rap1 signaling in zebrafish results in cyclopia. Cyclopia (fused eyes at the midline) was seen between 2 and 3 dpf in a small percentage of embryos injected with DN-Rap1 (15%, n = 94), SIPA1L1(CΔ) (7%, n = 115), or Rap1B MO (15%, n = 34). Cyclopia was seen in less than 1% of wild-type (n = 226) and GFP-injected (n = 111) controls.
(C) The ventral midline and neural plate are broadened in Rap1-deficient embryos. In situ analysis of the ventral midline by sonic hedgehog (shh) staining and the anterior boundary of the neural plate by distalless (dlx3) staining at the one- to three-somite stages revealed a broadening of the midline and neural plate in DN-Rap1- (34%, n = 29), SIPA1L1(CΔ)- (22%, n = 9), and Rap1B-MO- (42%, n = 24) injected embryos. Similar defects were seen in only 14% of uninjected controls (n = 77).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
DN-Rap1 and SIPA1L1(CΔ) Do Not Interfere with the β-Catenin Pathway
(A) DN-Rap1 and SIPA1L1(CΔ) do not block dorsal gene expression but cause an expanded field of expression of β-catenin target genes. (1 and 2) Uninjected control. (3 and 4) Wnt-8 + GFP; (5 and 6) Wnt-8 + DN-Rap1; (7 and 8) CKIɛ + GFP; (9 and 10) CKIɛ + DN-Rap1; and (11 and 12) CKIɛ + SIPA1L1(CΔ) mRNA were injected into a single ventral cell of four-cell stage embryos using the same quantities as in Figures 4A and 5B. In situ hybridization assays probing Goosecoid (Gsc) and Chordin (Chd) were performed on stage 10.5 embryos.
(B) DN-Rap1- and SIPA1L1(CΔ)-mediated gastrulation defects are not rescued by β-catenin. (i) Uninjected control. Xenopus embryos injected with mRNAs encoding β-catenin (100 pg) + GFP (250 pg) (ii); β-catenin + DN-Rap1 (100 pg) (iii); and β-catenin + SIPA1L1(CΔ) (250 pg) (iv) injection, photography, and scoring were performed as in Figure 4A and the scoring results are shown in the right panel.
(C) Schematic model of CKIɛ-mediated Wnt-8 pathway. Wnt-8 signaling activates CKIɛ (1; Swiatek et al., 2004), leading to phosphorylation and degradation of SIPA1L1 (2; see the result in Figure 2A) and accumulation of β-catenin (5; Gao et al., 2002). Degradation of SIPA1L1 results in the accumulation of GTP-Rap1 (3; see the result of Figure 2D), promoting convergent extension during gastrulation (4; see the results in Figures 4–6). In a separate pathway, stabilized β-catenin activates Lef1-TCF-dependent gene expression (5). Both Rap1-mediated convergent extension and β-catenin-regulated gene transcription contribute to the proper development of the body axis.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions