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Figure 1. Xenopus appl1 is expressed in the developing pancreas and SD of tadpole stage embryos. A-I: Whole mount in situ hybridization data for appl1 expression. A, B: Blastula and gastrula embryos bisected after whole mount in situ hybridization. C, D: Anterodorsal view. E: Lateral view. F: Dorsal view. G-I: Whole mount in situ hybridization on dissected whole guts. J: Western blot revealed temporal expression of appl1 during Xenopus embryogenesis. Beta-actin served as a protein loading control. K: RT-PCR analysis of appl1 temporal expression. UE, unfertilized egg. N, negative control. Ornithine decarboxylase (ODC) was used as the RNA loading control.
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Figure 2. Xenopus appl1 is required for gut development. A:Xenopus appl1 knockdown upon vegetal pole injection of 40 ng of Mo1 resulted in an edema phenotype that can be classified into three categories: severe edema, moderate edema, and normal. B: Edema phenotype caused by 40 ng of Mo1 can be rescued by co-injection of 1 ng of human APPL1 mRNA. Mo1 injection was repeated 5 times with 386 embryos injected in total. Co-injection was done 3 times with 274 embryos injected in total. C: Mo1 inhibited appl1 translation in a dose-dependent manner. Different doses (60, 40, and 20 ng) of Mo1 and 60 ng of MoC were injected into all four vegetal blastomeres of 8-cell-stage embryos and the injected embryos were subjected to Western blot analyses at stage 43. D: Western blot analysis confirmed that Mo1 could not inhibit human APPL1 translation in embryos co-injected with 40 ng of Mo1 and 1 ng of human APPL1 mRNA.
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Figure 3. Xenopus appl1 knockdown leads to a specific inhibition of pancreas and SD marker gene expression during tadpole stages of development. A: Whole mount in situ hybridization analysis revealed that XPDIp expression was significantly suppressed in Mo1-injected stage-42 embryos and inhibition levels were correlated with the severeness of the edema. B: Whole mount in situ hybridization analyses of XHex, XlHbox8, Foxa1, Ptf1a/p48, and XPDIp expression in Mo1, MoC, Mo1/APPL1, and Mo1/akt2-injected embryos. C:Insulin, insm1, Ptf1a/p48, and XlHbox8 expression at tail bud stage was not significantly altered upon Mo1 injection. The injection was done with 40 ng of MoC, 40 ng of Mo1, 40 ng of Mo1 plus 1 ng of human APPL1 mRNA, or 40 ng of Mo1 plus 1 ng of akt2 mRNA into all four vegetal blastomeres of 8-cell-stage embryos.Download figure to PowerPoint
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Figure 5. Xenopus akt2 shares a similar expression pattern with appl1 and can rescue the phenotype of appl1 knockdown. A: Whole mount in situ analysis indicates that akt2 is also expressed in pancreas and SD of tadpole-stage embryos (c–e). a, b: Lateral view. c–e: Dissected guts. B: RT-PCR analysis of temporal expression pattern of akt2. ODC was used as the RNA loading control. C: The edema phenotype caused by appl1 Mo1 was rescued by co-injecting 1 ng of akt2 mRNA with 40 ng of Mo1. The co-injection was repeated 3 times with 212 embryos injected in total. ME, moderate edema; N, normal; SE, severe edema. D: Western blot analyses revealed that the down-regulation of akt phosphorylation levels upon appl1 knockdown can be rescued by akt2. In this case, the injection was done at 8-cell stage and all eight blastomeres were injected with reagents indicated. The injected embryos were subjected to Western blot analyses when control siblings reached stage 41. Beta-actin was used as the protein-loading control.Download figure to PowerPoint
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Figure 6. Inhibition of akt activities via Wortmannin treatment leads to a phenotype that resembles appl1 knockdown effects. A: Lateral view of embryos that were treated with either DMSO or 100 nM Wortmannin from stage 30 to stage 42. B: Western blot data on embryos treated as described in A. C: TUNEL staining of embryos that were treated with either DMSO or 200 nM Wortmannin from stage 30 to stage 42. 1, 3: Dorsal view. 2, 4: Ventral view. D: Whole mount in situ hybridization data on embryos that were treated either with DMSO or 400 nM Wortmannin from stage 30 to stage 42. 1–6: Lateral view.Download figure to PowerPoint
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Supp. Fig. S1. Comparison of appl1, akt2, and appl2 expression in stage-33–40 embryos. The embryos cut transversally at the position of pancreas and SD primordia were subjected to whole mount in situ hybridization analysis
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Supp. Fig. S2. Spatial and temporal expression of appl2 in Xenopus embryos. A–I: Spatial expression of appl2 in Xenopus embryos revealed by whole mount in situ hybridization. A: Lateral view of a blastula embryo with signals mainly detected in the animal hemisphere. B: Bisected half of a gastrula embryo showing weak and ubiquitous expression. C: Anterodorsal view. D: Anterior view. E: Lateral view. F: Dorsal view. G–I: Dissected guts probed with appl2. In comparison to those observed for appl1 (Fig. 1G–I) and akt2 (Fig. 5A), only faint signals in the pancreas and SD were detected after a much longer period of color reaction incubation during the last step of whole mount in situ hybridization. J: RT-PCR analysis reveals the temporal expression of appl2. UE, unfertilized egg; N, negative control. ODC was used as the RNA loading control.
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Supp. Fig. S3. Dose-dependent induction of apoptosis in Xenopus embryos upon Wortmannin treatment. The embryos were treated with DMSO or 100–400 nM of Wortmannin from stages 30–42 and were subjected to TUNEL analysis at stage 42.
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Supp. Fig. S4. Dose-dependent inhibition of pancreas and SD marker gene expression in Xenopus embryos upon Wortmannin treatment. The embryos were treated with DMSO or 100–400 nM of Wortmannin from stages 30–42 and were subjected to whole mount in situ hybridization analysis at stage 42. Note that the duodenum expression domain of Foxa1 was nearly lost in 400-nM Wortmannin-treated embryos.
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Fig. 4. Xenopus appl1 knockdown leads to apoptosis. Ventral view of TUNEL staining of the embryos that were injected with 40 ng of MoC, 40 ng of Mo1, 40 ng of Mo1 plus 1 ng of human APPL1 mRNA, or 40 ng of Mo1 plus 1 ng of akt2 mRNA from vegetal pole at 8-cell stage. B: Dashed circle demarcates the region that is roughly equiva- lent to the pancreas and SD in a stage-42 control embryo. A: MoC injected. C: Human APPL1 rescue. D: Xenopus akt2 rescue.
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appl1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 7, mid-sagittal section, dorsal up.
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appl1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage11, mid-sagittal section, dorsal right.
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appl1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up.
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appl1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 21, anterior view, dorsal up.
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appl1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 4 , isolated gut tube, anterior left.
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appl1 (adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 32, lateral view, dorsal up, anterior left
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akt2 (v-akt murine thymoma viral oncogene homolog 2) gene expression in Xenopus laevis embryo, via in situ hybridization, NF stage 7 , lateral view, dorsal up.
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akt2 (v-akt murine thymoma viral oncogene homolog 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 42 , isolated gut tube, anterior left.
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foxa1 (forkhead box A1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 42, venrolateral view, anterior left.
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