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Fig. 6
Activity of XRP44X on microtubules during zebrafish segmentation. a, b Zebrafish blastomere cleavage at the 32 cell stage, F-actin in green (phalloidin), alpha tubulin in magenta and DNA with DAPI. a DMSO control, normal division with mitotic spindles (arrow). b XRP44X treated embryos, short microtubules, no mitotic spindle (arrowhead). Scale bar = 10 µm. c–e Embryos at 36 hpf, showing DMSO control, 2 µM XRP44X and 0.01 µM combretastatin A4 treatment. Xirp2a mRNA shows myotome boundaries with chevron pattern in DMSO (c; inset), ectopic xirp2a staining between faint and blurry boundaries with small molecule treatments (d, e; inset). f–h F-actin (phalloidin, green) in 20 ss embryos showing most recently formed boundaries (arrows). f In DMSO, thin straight boundary lines form between somites. With XRP44X g and combretastatin A4 h treatments, boundaries formed less clearly, and mature boundaries had gaps (asterisks). i–k During myogenesis, muscle fibers elongate (blue lines) to span the myotome i. With XRP44X and combretastatin A4, muscle fibers do not fully span the myotome j, k remaining partly rounded (arrowheads). l, m Myotomes of DMSO control and small molecule-treated embryos at 36 hpf. F-actin visualized with phalloidin (gray). Muscle fibers are well organized within each myotome in DMSO l, vertical myotome boundaries are highlighted as blue lines. m, n Small molecule-treated embryos showed loss of muscle fiber structure with round and short muscle fibers (arrowheads) and no clear myotome separation. XRP44X m treatment showed a higher level of disorganization than combretastatin A4 n. c–h, l–n scale bar = 50 µm, i–k scale bar = 20 µm. N = 30, 3 of 3 experiments showed representative phenotypes