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Fig. 2. Expression patterns of genes responsible for segmental patterning are altered in embryos lacking Mespo function. For all embryos, anterior is to the left. (A–K) One VMZ cell of four-cell-stage embryos injected with control MO (A, D, F, H and J), or Mespo MO (B, E, G, I and K) or Mespo MO plus 35 pg pCS–mMespo (C) and the embryos were fixed at neurula stage for in situ hybridization to detect the PSM marker gene expression: (A--C) PAPC, (D, E) Thy, (F, G) XDelta-1, (H, I) XBra, (J, K) Mespo. Bracket in panels B and E indicates the disruption of segmental expression of PAPC and Thy, respectively. Bracket in panel C indicates the rescue of PAPC expression by coinjecting Mespo MO with pCS–mMespo. Arrowhead in panel G indicates the disruption of segmental expression of XDelta-1. Arrowhead in panel I indicates the increase of XBra expression. Arrowhead in panel K indicates the increase of Mespo expression.
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Fig. 3. Wnt/β-catenin signaling in the PSM. (A–J) The neurula stage embryos were stained by whole-mount in situ hybridization for Xwnt8 (A, B, arrowhead), Dkk1 (C, D, arrow), Xwnt8 and Dkk1 (E, F, arrowhead indicates XWnt8 and arrow indicates DKK1), Xwnt8 and Thy (G, H, arrowhead indicates XWn8 and arrow indicates Thy), or Mespo and Thy (I, J, arrowhead indicates Mespo and arrow indicates Thy). Embryos in panels A, C, E, G and I are oriented with anterior to the left and shown in lateral views. Embryos in panels B, D, F, H and J show posterior–dorsal view. (K) Schematic diagram (adapted from Kim et al., 2000) illustrates gene expression patterns related to somite formation in the PSM of Xenopus embryos.
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Fig. 4. Wnt/β-catenin signaling regulates Mespo expression. For all embryos, anterior is to the left. Four-cell-stage embryos were injected in VMZ of one blastomere with lineage tracer pCS–LacZ as control (A, D), or together with pCS–Lef1–VP16 (B, E) or together with pCS–Lef1–EnR (C, F). At stage 19, whole mount in situ hybridization was performed for Mespo (A--C) or Paraxis (D--F), as indicated. Arrowhead in panel B shows the increase of Mespo expression. Arrowhead in panel C shows the decrease of Mespo expression.
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Fig. 5. The LEF/TCF binding site is responsible for the expression of Mespo. (A) Schematic representation of Mespo promoters from various species with a conserved LEF/TCF binding site (red). The position of potential LEF/TCF binding site (box) in Xenopus Mespo 5′-upstream sequence is indicated. (B–D) Fold induction of pCS–Lef1–VP16 in pGL3basic and p-4317Luc reporter plasmids (B), luciferase activities of p-4317Luc and p-4317mLuc reporter plasmids (C) or fold induction of pCS–Lef1–VP16 in p-4317Luc and p-4317mLuc reporter plasmids (D) in animal cap assays. Plasmids were injected into the animal pole at the four-cell stage. Animal caps were dissected at stage 8.5 and luciferase activities were measured at stage 12.5. RLU, relative light units. (E) Luciferase activities of the indicated plasmids injected into both VMZ cells at four-cell stage and measured at stage 19. RLU, relative light units. (F) Expression of endogenous Mespo mRNA at the tailbud stage. (G–I) Expression of GFP mRNA in p-4317GFP transgenic embryos (G) or in p-4317mGFP transgenic embryos (H) at the tailbud stage. The percentages of transgenic embryos that show the PSM expression (purple), non-specific expression (brown), and no detected signal (white) are shown in panel I.
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Fig. 7. PI3-K/AKT signaling regulates Mespo expression. (A) Hatched lines indicate the three caudal regions (C, R, and S) used for western blot of Akt and phosphorylated Akt. (B) Western blot of Akt and phosphorylated Akt in different regions indicated in panel A. (C) Western blot of phosphorylated Akt in the caudal PSM equivalent to region C (illustrated in panel A) treated with either LY294002 or DMSO. (D–G) The neurula stage embryos were treated with either DMSO (D, F), or LY294002 (E, G) for 2 h, and then stained for Mespo and Paraxis as indicated. (H) RT–PCR of Mespo mRNA in the caudal PSM equivalent to region C (illustrated in panel A) treated with either DMSO or LY294002. The caudal PSM were dissected at stage 23 and treated with drugs for 2 h, then harvested for RT–PCR.
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Fig. 8. Interaction between PI3-K/AKT and Wnt/β-catenin signaling in PSM. (A) Western blot of β-catenin in the nucleus and cytosol of the caudal PSM cells from embryos treated with either DMSO or LY294002 for 2 h. (B) Western blot of β-catenin, phosphorylated β-catenin, Akt and phosphorylated Akt in the caudal PSM cells treated with DMSO for 2.5 h, or LY294002 for 1.5 h or 2.5 h. (C–E) Four-cell-stage embryos were injected in VMZ of one cell with pCS–LacZ as control (C) or together with pCS–Lef1–VP16 (D, E). At the neurula stage, injected embryos were treated with DMSO (C, D) or LY294002 (E) for 2 h, then fixed, stained with X-gal to reveal the tracer (light blue) and analyzed by whole-mount in situ hybridization for Mespo. Arrow in panel D indicates the increase of Mespo expression in the injected side and arrowhead indicates the expression of Mespo in the uninjected side. Arrow in panel E indicates the increased Mespo expression in the injected side and arrowhead indicates the decrease of Mespo expression in the uninjected side.
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Supplemental Fig. 1. PCSMespo rescued the phenotypical effects of Mespo MO. (A, B) All embryos were injected with Mespo MO plus 15 pg pCSacZ (A) or together with 35 pg pCSMespo DNA (B). Injection of pCSMespo effectively rescued the phenotypic effects of Mespo MO in 85% (N = 100) of the injected embryos (compare A and B).
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Supplemental Fig. 2. Wnt/β-catenin signaling regulates Mespo expression. (A, B) Enhancing Wnt/β-catenin signaling activity increases Mespo expression. Four-cell-stage embryos were injected in VMZ of one cell with pCSacZ (A) or together with pCS-Beta-catenin DNA (B). While the expression of Mespo in the pCSacZ injected side in stage 19 embryos is the same as the uninjected side (A), the expression of Mespo in the pCS-beta-catenin injected side is increased (B). Arrowhead in (B) indicates the increase of Mespo expression and arrow indicates the expression of Mespo in the uninjected side. For all embryos, anterior is to the left. (C, D) Overexpression of Lef1-EnR mRNA decreases the expression of Mespo. Four-cell-stage embryos were injected in VMZ with 50 pg LacZ mRNA (C) or together with 400 pg Lef1nR mRNA (D). (C) Expression of Mespo at stage 10. No significant difference can be found between the uninjected side and injected side. (D) In Lef1nR mRNA injected side, Mespo expression is absent (80%, N = 50). Arrowhead in (D) indicates the decrease of Mespo expression. All the embryos are vegetal view with dorsal up.
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Fig. 1. Antisense MO against Mespo depletes Mespo protein and causes somite formation defects. (A) Sequence of Mespo MO aligned with the 5′-untranslated region (UTR) of Mespo RNA. (B) In vitro transcription/translation of Mespo is inhibited by Mespo MO. Arrow indicates the band corresponding to Mespo protein. (C, D) Whole mount immunostaining with 12/101, a muscle-specific antibody, shows that the segmental pattern of somites in Mespo MO-injected embryos (D) is disrupted and the somite size is irregular (compared with control MO-injected embryo in panel C). Bracket in panel D marks the malformed somites. (E) Embryos injected with Mespo MO (F, H) or control MO (E, G) stained with 12/101, then sectioned longitudinally and labeled with DAPI. Panels E, G and Panels F, H are the same view of one embryo, respectively. Embryos injected with Mespo MO panel F, H show the disorganization of segmental pattern, based on myotomal morphology (panel F, compared with panel E) and the arrangement of somatic nuclei panel H, compared with G). Bracket in panel H marks the malformed somites.
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dkk1 (dickkopf WNT signaling pathway inhibitor 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
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