THN progenitors initiate axonogenesis from their leading process independent of centrosome proximity. (A) Lateral view of the cerebellar anlage of an ∼42-hpf Tg(atoh1a:Gal4TA4)^hzm2 transgenic zebrafish embryo injected with Medusa vector M1. An axon-like protrusion (white asterisk) has formed at the time when the centrosome (white arrow) is still homing toward the soma. (B) Lateral view of a Tg(atoh1a:Gal4TA4)^hzm2 x Tg(4xUAS:GFP)^hzm3 transgenic zebrafish embryo at 42 hpf. GFP-expressing cells are visualized by anti-GFP immunostaining (green) and acetylated microtubules by anti-acetylated tubulin immunostaining (red). (C) Enlargement of boxed area in B. Arrows indicate acetylated microtubules present in GFP-expressing THN progenitors, indicating the presence of axons by 42 hpf. (D–F) Lateral view of the cerebellum of a 40-hpf Tg(atoh1a:Gal4TA4)^hzm2 transgenic zebrafish embryo injected with Medusa vector M1. (D) A THN progenitor (white asterisk) extends a process, the presumptive axon with a growth cone–like structure (red arrow), while the centrosome (white arrow) starts to translocate toward the soma. (E and F) The axon-like process elongates while the centrosome is moving toward the soma and is still far removed from the site of axonogenesis. These findings suggest that the site of axon formation in THN neurons is independent of a proximally positioned centrosome. Images in D–F are taken from Video 9. MHB, midbrain–hindbrain boundary; URL, upper rhombic lip.