|
Fig. 3 Wnt5b regulates tubulin protein levels and microtubule dynamics at the MHB. (A) Representative western blot for α-tubulin and β-tubulin, with β-actin as the control, comparing control MO- and wnt5b MO-injected embryos. Head tissue protein was analyzed. (B) Quantification of at least three independent western blot (WB) experiments; α-tubulin and β-tubulin levels were normalized to β-actin. Data are represented as mean±s.d. (C,D) Representative live confocal images showing 10 μm average intensity projections at 22-24 ss of wild-type embryos co-injected with EMTB-GFP, memCherry, and control MO (C) or wnt5b MO (D). (E) Quantification of the normalized MHBC EMTB-GFP intensity in control versus wnt5b morphants (Fig. S4A-C). Boxplots indicate the 25th and 75th percentiles and the median. Three independent experiments are represented. Control MO, n=10; wnt5b MO, n=7. Scale bars: 20 μm. (F,G) Representative confocal images of β-tubulin immunostaining showing 10 μm average intensity projections at 24 ss of wild-type embryos injected with either control MO (F) or wnt5b MO (G). (H) Quantification of the normalized basal MHBC intensity in control versus wnt5b morphants. Basal MHBC intensity was divided by the intensity in the middle of the cell (see Fig. S4D-F). Boxplots indicate the 25th and 75th percentiles and the median. Three independent experiments are represented. Control MO, n=7; wnt5b MO, n=6. Scale bars: 10 μm. (I-K) Quantification of control versus wnt5b morphants for EB3-GFP comet speed (I), EB3-GFP comet number (J) and EB3-GFP comet size (K). See also Movies 1-4. Boxplots indicate the 25th and 75th percentiles and the median. Three independent experiments are represented. In J,K, data are represented as mean±s.e.m. EB3-GFP comet speed: control MO, n=11; wnt5b MO, n=7. EB3-GFP comet number and comet size: control MO, n=11; wnt5b MO, n=11. *P<0.05, ***P<0.005. Arrowheads indicate MHBC.