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FIGURE 1:. TACC3 protein is expressed within embryonic neuronal growth cones and promotes axon outgrowth. (A–F) Immunostaining with antibodies to TACC3 (A) and tubulin (B) in cultured embryonic neural crest cells confirm that TACC3 is enriched at the centrosome (C, A′–C′), in addition to being present in puncta throughout the cell. (D–F) TACC3 antibody also strongly stains growth cones. Scale bar, 5 μm. (G) Western blot showing that a morpholino (MO) targeted against TACC3 results in 50% knockdown (KD) by 2 d postfertilization. Bar graph depicts densitometry of blot shown; however, similar results were obtained with >20 individual Western blots (unpublished data). (H–I) Axon outgrowth parameters after 24 h in culture were quantified in control and TACC3 KD conditions. (H) The numbers of axons per neural tube explant were counted from three independent experiments. (I) The distance from explant to growth cone was measured to calculate the length of axons from three independent experiments. (J–K) Representative phase contrast microscopy images of axons from control and TACC3 KD. (L) Western blot showing that TACC3 levels are increased (overexpressed [OE]) 2 d after injecting embryos with TACC3 mRNA. Bar graph depicts densitometry of blot shown; however, similar results were obtained with >20 individual Western blots (unpublished data). Note that the TACC3 blot in L was exposed for a shorter amount of time than the one in G, which explains the fainter control band in L. (M, N) Axon outgrowth parameters after 18 h in culture were quantified in control and TACC3 OE conditions. (O–P) Representative phase contrast microscopy images of axons from control and TACC3 OE. Box-and-whisker plots indicate the mean (diamond), median, extrema, and quartiles. An unpaired t test was performed to assess significance between conditions. **p < 0.01, ***p < 0.001; n.s., not significant. Bar, 50 μm (J, K, O, P). |
