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Graphical Abstract
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Fig. 2. Gli1 co-localizes with Zyxin in Xenopus laevis fibroblasts. A�C. Cells expressing Myc-Gli1 (A), Zyxin-EGFP (B) or EGFP alone. Here and below, Myc-Gli1 was revealed by Texas Red labeled anti-Myc antibody; n�nucleus; scale bar: 5 μm. D. A cell co-transfected with plasmids encoding the full-length Myc-Gli1and Zyxin-EGFP. White arrows indicate true yellow color, which confirm tight co-localization of two proteins. E. No co-localization (no true yellow color) of mutant Gli1, lacking zinc fingers, and Zyxin is seen in the cell co-transfected with pMyc-mutGli1and pZyxin-EGFP. F. Nuclear to cytoplasmic fluorescence density ratio (DFR) in cells transformed with Myc-Gli1, Myc-mutGli1and EGFP-Zyxin alone or together. DFR was calculated for 200 cells of each type in two independent experiments.
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Fig. 3. Shh, Gli1, Ptc2 and Zyxin expression in CNS of the Xenopus laevis embryo A�H. In situ hybridization with probes to Shh, Gli1, Ptc2 and Zyxin on successive sections of two embryos, at stage 15 (A, C, E and G) and stage 27 (B, D, F and H). I and I′. The midneurula (stage 15) transgenic embryo expressing Zyxin-EGFP under the control of CMV promoter. Zyxin-EGFP signal is concentrated (arrowheads) along apical membranes of the flour plate cells. Dorsal view, anterior to the top. J and K. At the mid (stage 14) and late (stage 18) neurula stage transgenic embryos, Zyxin-EGFP signal is concentrated (arrowheads) along membranes of the floor plate cells. Fp�floor plae, lnp�lateral neural plate, ml and vl�mantle and ventricle layers of the neural tube respectively, hy�hypochord, np�neural plate, not�notochord, s�somite. Scale bar everywhere is 40 μm.
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Fig. 5. Expression of Shh genetic targets in the neural plate of embryos injected by AntiZyxin Mo. A�F. Almost complete inhibition of Pax6, Dbx1 and Olig4 expression (normally inhibited by Shh signaling) in areas populated by cells containing antiZyxin Mo (see A′�F′ for localization of FLD tracer). Control embryos were injected with misantiZyxin Mo. All embryos are shown from dorsal side, anterior to the top. Arrows indicate sites of the expression inhibition. Transverse sections of embryos at the level indicated by dashed line on A�F are shown on A′�F′. G�L. A weak expansion of the Foxa2, Gli1 and Ptc2 expression (normally activated by high Shh signaling) in areas populated by cells containing antiZyxin Mo (see G′�L′ for localization of FLD tracer). Yellow stripes indicate borders of expression domains. Transverse sections of embryos at the level indicated by dashed line on G�L are shown on G′�L′.
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Fig. 6. Influence of Zyxin on Shh/Gli1-responsive genes in embryos with overexpressed Shh. 8-cells embryos were injected into the right dorsal blastomere with the following amount of mRNAs indicated in the top line: Zyxin�150 pg/embryo; Shh�15 pg/embryo; Zyxin-δ2LIM�150 pg/embryo, along with the living tracer, FLD. After in situ hybridization of embryos at stage 25 with probes to Pax6 (A�D), Dbx1 (F�I), Nkx2.2 (K�N) and Foxa2 (P�S), sagittal projections of expression domains of each gene were measured on the left (control) and right (injected) sides of the neural tube and percentage change of expression on the injected side (δ) was calculated (see diagrams in E and O). Mean value and standard deviation of the expression domain changes for all embryos injected by the same set of mRNAs are shown on E, J, O and T by columnar diagrams. Red bars on photos indicate borders of the expression domains on the injected and non-injected sides of embryos. Dotted line indicate border of the neural tube. Red dotted lines on schematic representation of neural tube section on E indicates normal positions of the ventral borders of the expression domains of Pax6, n�number of embryos analyzed in each case.
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Fig. 7. Influence of Zyxin on Gli1/Shh-responsive genes in embryos with overexpressed Gli1. Embryos were injected in the same way as that in Fig. 6, except that 10 pg/embryo of Gli1 mRNA was injected instead of Shh mRNA. At stage 27, embryos were cut into successive transverse vibratome sections in the hindbrain region and four sections of each embryo were hybridized individually with probes to Pax6 (A�C), Dbx1 (E�G), Nkx2.2 (I�K) and Foxa2 (M�O). For abbreviations, see Fig. 6.
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Fig. 8. Zyxin interferes with Gli1 binding to its target promoter. A. Luciferase reporter assay of Zyxin effects upon Gli1, Gli2 and Gli3 activity. 8�3′Gli-BSLuc reporter (30 pg/embryo) was mixed with the reference pCMV-β-GAL plasmid (30 pg/embryo), indicated mRNAs (Gli1-3�70 pg/embryo; Zyxin, NLS-Zyxin, EnR-Zyxin, VP16-Zyxin�150 pg/embryo each); and injected into embryos at 2�4 blastomeres stage. All experiments were done in triplicates. See Fig. 1 and Martynova et al. (2008) for principal protein structures. B. EMSA demonstrating reduced binding of Gli1 to its DNA target site in the presence of Zyxin.
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Figure S1. LIM domains�containing part of Zyxin binds to the zing-finger region�containing fragments of Gli1 in IP assay. A. Principal scheme of Gli1 fragments used for IP assay with FLAG-ZyxinLIM1-3. Gli1 fragments demonstrating binding to FLAG-ZyxinLIM1-3 (see original results on B) are marked by �+� or �−�. B. IP assay of abilities of Myc-tagged Gli1 fragments shown on A to bind to FLAG-ZyxinLIM1-3 in extracts of Xenopus embryos injected with the corresponding mRNAs. C. Comparison of FLAG-ZyxinLIM1-3 and FLAG-ZyxinδLIM2 (FLAG-ZyxinLIM1-3 lacking LIM2) in co-immunoprecipitation with Myc-N-Gli1.
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Figure S2. Injection of Xenopus laevis embryos with 0.1 mM of AntiZyxin MO (by 2 nl into each blastomere at 2-cells stage) resulted in a diminishing of the overall amount of endogenous Zyxin at the midneurula stage (stage 15).
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Figure S3. Transverse vibratome section of the transgenic embryo at the tailbud stage (stage 22) expressing Zyxin-EGFP under the control of CMV promoter. A. EGFP channel. B. DAPI channel. Nt�neural tube, s�somite, not�notochord. Dorsal to the top.
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Figure S4. Rescue of the anti-sense Mo-induced effects of the endogenousZyxinmRNA translation by co-injection ofZyxinmRNA lacking the Mo target site. A. Unilateral injection of Zyxin mRNA (100 pg/embryo) elicits expansion (arrow) of Pax6 expression on the injected side. B. In contrast, injection of anti-Zyxin Mo leads to inhibition of Pax6 expression (arrow). C. Co-injection with anti-Zyxin Mo of Zyxin-misMo mRNA (100 pg/embryo) partially rescues (arrow) the Mo effect. D and E. Co-injection with anti-Zyxin Mo of Zyxin-misMo mRNA (100 pg/embryo) partially rescues normal phenotype of embryos. Normal and abnormal embryos are indicated by �+� and �−�, respectively.
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