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FIGURE 1:. Embryonic FAK expression and MO knockdown in Xenopus embryos. (A) In situ hybridization to FAK mRNA in neurula embryos. Embryos are viewed dorsally and oriented anterior (top), posterior (bottom); left, stage 17; right, stage 20. (B) Western analysis shows that FAK MO and SB FAK MO effectively reduce FAK protein levels in morphant embryos (compare lanes lane 1 and 2, and 5 and 6, respectively) and AC explants (compare lanes 3 and 4). Approximately 30 μg of total protein was loaded per lane. Loading per sample is determined by α-tubulin protein.
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FIGURE 2:. FAK morphant embryos are anteriorized. (A) Late neurula-stage control embryo. (B) Embryos were injected with FAK MO (60 ng) at the one-cell stage. FAK MO–injected embryos are anteriorized, having a shortened A-P axis and open neural folds in 100% (n = 23) of the embryos. (C) The splice-blocking FAK MO (20 ng)–injected embryos had the same phenotype as in B in 96% (n = 26) of the embryos. (D) TO-PRO-3 iodide nuclear staining of neural tube cross sections in FAK morphant embryos. A transverse view, with dorsal oriented to the top and ventral to the bottom. FAK MO (30 ng) was injected into one blastomere at the two-cell stage. At neurula stages, embryos are transversely sectioned and nuclei stained with TO-PRO-3 iodide. The neural folds are visually analyzed. The arrow indicates the dorsal midline. The injected side is on the left, indicated by a star. The elevation of the neural folds on the injected side (left) is perturbed in comparison to the uninjected control side (right). (E) Control uninjected sibling embryo treated as in D. (F) A lateral view of a control tadpole-stage embryo: oriented anterior to left, posterior to right. Normal phenotypes in 89% (n = 38) of the embryos. (G) In the FAK MO (30 ng)–injected embryos, the A-P axis is truncated, the tail is shortened, and the cement gland is expanded. Anteriorized phenotypes in 97% (n = 31) of the embryos.
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FIGURE 3:. The FAK MO and the splice FAK MO perturb posterior neural marker expression in neurula-stage embryos. (A) Embryos (lane 2) were injected at the one-cell stage with the FAK MO (60 ng, lane 3), splice-blocking FAK MOs (18 ng each, lane 4), or both types (lane 5). At early neurula stages, expression of the posterior neural homeobox marker genes HoxD1, Meis3, Gbx2, and HoxA2 was examined by sqRT-PCR. In all experiments, RNA is isolated from pools of 5–10 embryos per group. EF1a serves as a positive control for RNA loading in all shown experiments. (B) RNA from sibling embryos shown in A was isolated at late neurula stages. Expression of the posterior neural homeobox marker genes Krox20, HoxB3, HoxB9, and N-tubulin was examined by sqRT-PCR. Expression of the anterior forebrain marker XAnf1 was also examined. Both MOs inhibited posterior neural marker expression while up-regulating anterior marker expression. (C) By in situ hybridization, FAK and Wnt3a morphant phenotypes are compared. Three different posterior cell types were analyzed by the following probes: Krox20 (a–c, hindbrain), N-tubulin (d–f, primary neuron) and FoxD3 (g–i, neural crest). Embryos were injected at the one-cell stage with the FAK MO (60 ng; b, e, h) or the Wnt3a MO (30 ng; c, f, i). Arrowheads (a–c) mark the r5 Krox20 expression band, and full arrows (d–f) mark the trigeminal (V cranial) nerve expression of N-tubulin. Embryos are at late neurula stages, viewed dorsally; anterior is on top. (a) Krox20 expression is normal in all embryos (n = 10). (b) Krox20 expression is perturbed in 90% of the FAK morphant embryos (n = 10). (c) Krox20 expression is perturbed in all of the Wnt3a morphant embryos (n = 14). (d) N-tubulin expression is normal in all embryos (n = 10). (e) N-tubulin expression is perturbed in all of the FAK morphant embryos (n = 7). (f) N-tubulin expression is perturbed in all of the Wnt3a morphant embryos (n = 17). (g) FoxD3 expression is normal in all embryos (n = 10). (h) FoxD3 expression is at normal levels but morphologically perturbed in 75% of the FAK morphant embryos (n = 12). (i) FoxD3 expression is at normal levels but morphologically perturbed in all of the Wnt3a morphant embryos (n = 16).
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FIGURE 4:. The FAK morphant phenotype is rescued by overexpression of FAK mRNA. (A) Posterior neural marker expression is rescued by ectopic FAK in morphant embryos. Embryos were injected with the FAK MO (50 ng) at the one-cell stage, and then full-length X. laevis FAK–encoding mRNA was injected at either the one-cell stage (80 pg) or the eight-cell stage, where RNA was injected into the upper animal tier of four blastomeres (12.5 pg/blastomere) fated for ectoderm. Total RNA was isolated at late neurula stages from pools of six embryos, and sqRT-PCR was performed to the posterior neural Krox20, HoxB9, Wnt3a, and N-tubulin marker genes. (B) Morphological rescue of neural folding defects in FAK morphant embryos. Embryos were separately injected with FAK MO and/or FAK mRNA at the one-cell or eight-cell stage as described for A, except that an additional embryo group was injected with 110 pg of FAK mRNA at the one-cell stage. Embryos were analyzed at late neurula stages: (a) 88% of the control embryos had normal neural folding (n = 34), (b–d) 92% of the FAK-expressing embryos had normal neural folding (n = 77), (e) 67% of the FAK morphant embryos had abnormal neural folding (n = 30), (f) 76% of the FAK MO + FAK (80 pg)–expressing embryos had rescued neural folding (n = 34), (g) 75% of the FAK MO + FAK (110 pg)–expressing embryos had rescued neural folding (n = 36), and (h) 56% of the FAK MO + FAK (12.5 pg/blastomere)–expressing embryos had rescued neural folding (n = 39).
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FIGURE 5:. FAK protein knockdown inhibits posterior neural induction by the FoxD1 protein. (A) One-cell-stage embryos were injected separately into the animal hemisphere with the FAK MO (60 ng) and/or 25 pg of FoxD1 mRNA. Animal cap (AC) explants were removed at blastula stages 8 to 9. Explants and embryos were cultured to neurula stages, and total RNA was isolated from pools of eighteen AC explants in each group and five control embryos (CE). sqRT-PCR analysis was performed with the markers XAnf1, otx2, HoxB9, Eph2a, Wnt3a, N-tubulin, and EF1α. sqRT-PCR was performed on total RNA isolated from normal embryos. (B) One-cell-stage embryos were injected separately into the animal hemisphere with the FAK MO (50 ng) and/or of FoxD1 (25 pg) mRNA. For rescue, FAK (80 pg) mRNA was injected. AC explants were cultured and RNA isolated as described in A. sqRT-PCR analysis was performed with the markers Wnt3a, HoxB9, Eph2a, Wnt3a, NCAM, and EF1α.
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FIGURE 6:. Comparison of posterior neural marker gene expression patterns in Dkk1-expressing and FAK morphant embryos. Embryos at the one-cell stage were injected with either FAK MO (60 ng) or Dkk1 (50 pg) mRNA. (A) In situ hybridization was performed at early neurula stages for two Hox genes, HoxD1 and HoxA2. Embryos are viewed dorsally, with anterior at the top: (a) 100% normal HoxD1 expression (n = 15), (b) 28% disrupted HoxD1 expression (n = 18), (c) 100% disrupted HoxD1 expression (n = 20), (d) 100%, normal HoxA2 expression (n = 15), (e) 100% disrupted HoxA2 expression (n = 18), and (f) 100% disrupted HoxA2 expression (n = 15). (B) In situ hybridization was performed at late neurula stages to N-tubulin (primary neuron) En2 (white arrow; midbrain–hindbrain junction), HoxB3 (black arrow; r5/6), and Slug (neural crest). Embryos are viewed dorsally, anterior at the top: (a) 100% normal n-tubulin expression (n = 15), (b) 100% disrupted n-tubulin expression (n = 18), (c) 100% disrupted n-tubulin expression (n = 9), (d) 100% normal En2/Hoxb3 expression (n = 15), (e) 95% disrupted En2/Hoxb3expression (n = 19), (f) 100% disrupted En2/Hoxb3 expression (n = 18), (g) 100% normal Slug expression (n = 15), (h) 83% disrupted but not reduced Slug expression (n = 18), and (i) 100% disrupted and reduced slug expression (n = 17).
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FIGURE 7:. FAK protein modulates Wnt activity. (A) The FAK MO decreases canonical Wnt signaling transcriptional activity. Embryos were injected with 25 pg of the 3X-TCF-luciferase reporter vector and 60 ng of FAK MO. The graph represents five different experiments at stages 11.5–13. In each experiment, a pool of five embryos was lysed per group. Luciferase activity was normalized to protein levels in control embryo extracts and was set at 100%. (B) The FAK MO (60 ng) down-regulates Wnt3a expression. In situ hybridization to Wnt3a was performed at late neurula stages. (a) Normal Wnt3a expression appears in 86% (n = 14) of the control embryos. (b) Wnt3a expression is eliminated in 95% of the FAK morphant embryos (n = 20). (C) Wnt3a and Wnt8 gene expression was compared from early to late neurula stages by sqRT-PCR in embryos injected with the FAK MO (60 ng). In FAK morphants Wnt3a expression is strongly inhibited at all the stages, whereas Wnt8 expression is normal. (D) Embryos were injected with the FAK MO (60 ng; lane 3), the splice-blocking MO (18 ng; lane 4), or both MOs (lane 5). sqRT-PCR to Wnt3a was performed at the indicated stages.
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FIGURE 8:. The FAK morphant phenotype is rescued by zygotic mouse Wnt3 ectopic expression. (A) Morphological rescue of neural folding defects in FAK morphant embryos. At the one-cell stage, embryos were separately injected with FAK MO (30 ng) and/or the mWnt3 expression vector driven by the CMV promoter (60 pg). Embryos were analyzed at late neurula stages. (a) All control embryos show normal neural folding (n = 27), (b) all FAK morphant embryos had perturbed neural folding (n = 32), (c) all posteriorized mWnt3-expressing embryos had normal neural folding (b = 16), and (d) FAK MO + mWnt3–expressing embryos had rescued neural folding in 94% of the embryos (n = 16). (B) Posterior neural marker expression is rescued by ectopic mWnt3 in FAK morphant embryos. Embryos were injected at the one-cell stage with FAK MO (30 ng; lane 3), mWnt3 expression vector (50 pg; lane 5), or both (lane 4). As determined by sqRT-PCR, inhibition of hindbrain/spinal cord (Krox20, HoxB3, HoxB9) and primary neuron marker (NeuroD, N-tub) expression, as well as the increase in the anterior XAnf1 marker (forebrain), in FAK morphants is rescued by ectopic mWnt3. C. Wnt3 suppresses expanded XAnf1 expression in FAK morphants. At the one-cell stage, embryos were separately injected with FAK MO (50 ng) and/or the mWnt3 expression vector (60 pg). In situ hybridization to XAnf1 was performed at late neurula stages. (a) Normal XAnf1 expression in control embryos (n = 68). (b) In FAK morphants, XAnf1 expression is strongly expanded in 95% of the embryos (n = 64). (c) In mWnt3-expressing embryos, XAnf1 expression is eliminated in 80% of the embryos (n = 81). In rescued, FAK MO/mWnt3–injected embryos (n = 77), only 26% have expanded XAnf1 expression (unpublished data), 22% have normal XAnf1 expression (d), 25% have slightly expanded expression (e), and 21% have no expression (f).
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FIGURE 9:. Depletion of FAK protein in human MCF-7 breast cancer cells reduces Wnt3 gene expression. (A) sqRT-PCR was performed on total RNA isolated from control siRNA-transfected, empty vector–transfected, and FAK siRNA–transfected human MCF-7 cells. Gene expression analysis was carried out for the human Wnt7a, Wnt3, Wnt3a, and GAPDH (positive control) genes. (B) Western analysis compares Wnt3 protein levels in two separately transfected FAK siRNA cell lines in comparison to a control siRNA. FAK siRNA effectively reduce Wnt3 protein levels vs. the control. Approximately 30 μg of total protein was loaded per lane. Loading per sample is determined by β-actin protein.
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