Colchicine and blebbistatin inhibit THN migration (related to Fig. 3 ). (A) Normal development of 29-hpf embryos. Scale bar, 200 µm. (B) The application of 5 mg/ml colchicine at 29 hpf leads to spine curvature after 5 h. Scale bar, 200 µm. (C) Colchicine induces cell rounding, first apparent in mitotic cells at the URL (arrows). Later, all cells begin to round and the tissue collapses without the formation of THN clusters at the ventral end of the MHB (13 hpt). THN cluster formation is also absent in blebbistatin-treated embryos, although cells with two nuclei (arrowheads) increase over time. Scale bar, 20 µm. (D) THNs treated with colchicine are able to form small, transient protrusions. Colors indicate two examples. Dots label the cell centers, arrowheads point toward protrusions. Elapsed time in minutes. Scale bar, 5 µm. (E) Lower concentrations of colchicine delay the onset of complete MT depolymerization, as indicated by the presence of Map4MTB-positive structures in many THNs after 9 hpt. At 13 hpt, most of these structures have been lost and extensive cell rounding with tissue collapse is apparent. Scale bar, 20 µm. (F) Tracking THNs migrating in the 9–13-hpt window of exposure to lower concentrations of colchicine reveals that incomplete loss of MTs already reduces THN motility. n = 24 embryos/145 tracks for control, n = 4 embryos/92 tracks for 1 mg/ml colchicine, n = 4 embryos/54 tracks for 0.5 mg/ml colchicine; P = 4.58 × 10−8 control/1 mg/ml colchicine, and P = 0.046 control/0.5 mg/ml colchicine. MHB, solid line; URL, dotted line. Boxes in graphs represent 25–75% of all values and whiskers 1.5 times the quartile. Median is shown as a horizontal bar and mean as a square box. Significance level in Kruskal–Wallis ANOVA: *, P < 0.05; ***, P < 0.001.
